Cyclic disulfide-modified phosphate-based oligonucleotide prodrugs

Abstract

The present invention relates to a compound of formula (I): [Cyclic disulfide moiety]-[Phosphorus coupling group] (I) The present invention relates to compounds comprising the structure of: The present invention has the structure JPEG2024501857000158.jpg38160. The present invention also relates to oligonucleotides comprising one or more compounds comprising the structure of formula (I), wherein at least one [phosphorus coupling group] comprises a nucleoside or oligonucleotide. The present invention also relates to pharmaceutical compositions comprising the oligonucleotides described herein and methods of reducing or inhibiting expression of a target gene by administering to a subject a therapeutically effective amount of the oligonucleotides described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 132,535, filed December 31, 2020; and U.S. Provisional Application No. 63 / 287,833, filed December 9, 2021, both of which are hereby incorporated by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates generally to the field of modified phosphate-based oligonucleotide prodrugs. [Background technology]

[0003] background Phosphate esters are key intermediates in nucleotide formation and assembly into RNA and DNA. Within cells, the phosphate group generally serves as a controllable leaving group. Phosphate esters are charged at physiological pH, which facilitates binding of the phosphate ester to the enzyme active site. However, for the phosphate ester to bind to the enzyme, it must first penetrate the membrane to access the enzyme, as charged molecules can have difficulty crossing the cell membrane except by endocytosis. This limitation can be alleviated for compounds with larger, more lipophilic substituents.

[0004] Alternatively, a prodrug approach has been investigated, which temporarily masks any negative charge on the phosphate ester of an oligonucleotide at physiological pH. A prodrug is a drug that is administered in an inactive or significantly less active form and undergoes chemical or enzymatic conversion in vivo under various stimuli to produce the active parent drug. The prodrug approach, which masks the negative charge on the phosphate group of an oligonucleotide with a cell-cleavable protecting / masking group, may offer several advantages over its non-protected counterpart, including, for example, enhanced cell penetration and avoidance or minimization of degradation in serum due to cell sequestration.

[0005] However, the prodrug approach still poses considerable challenges, particularly because it is difficult to select the best masking group. For example, cellular cleavage of the protecting group can often produce products that are considered undesirable or even toxic. Furthermore, the protecting group must strike a balance between allowing intestinal absorption and cleavage in the blood or target cells. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a continuing need for new and improved modified phosphate prodrugs that mask the internucleotide phosphate linkages of oligonucleotides to produce effective and efficient oligonucleotide-based drugs for efficient in vivo delivery of oligonucleotides and improved in vivo efficacy. [Means for solving the problem]

[0007] overview One embodiment of the present invention is a compound of formula (I): [Cyclic disulfide moiety]-[phosphorus coupling group] (I) The present invention relates to a compound comprising the structure: [ka] In these formulas, R1 is O or S and is attached to the P atom of the [phosphorus coupling group]; R2, R4, R6, R7, R8 and R9 are each independently H, halo, OR 13 or alkylene-OR 13 , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R 14 )(R 15 )(R 16 ) or alkylene-C(R 14 )(R 15 )(R 16), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R sub optionally substituted with a group; R3 and R5 are each independently H, halo, OR 13 or alkylene-OR 13 , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R 14 )(R 15 )(R 16 ) or alkylene-C(R 14 )(R 15 )(R 16 ), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R sub or R3 and R5 together with the adjacent carbon atom and two sulfur atoms form a second ring; G is O, N(R'), S or C(R 14 )(R 15 ) and; n is an integer from 0 to 6; R 13 is independently at each occurrence H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, each of which optionally contains one or more R sub optionally substituted with a group; R 14 , R 15 and R 16 are each independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, N(R')(R"); R′ and R″ are each independently H, alkyl, alkenyl, alkynyl, aryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, each of which optionally contains one or more R sub and R sub is independently at each occurrence halo, haloalkyl, alkyl, alkaryl, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, arylcarbonyl, acyloxy, cyano, or ureido.

[0008] In one embodiment, in the [cyclic disulfide moiety]: R1 is O; G is CH2; n is 0 or 1; R2, R4, R6, R7, R8 and R9 are each independently H, halo, OR 13 or C1-C6 alkylene-OR 13 , N(R')(R") or C1-C6 alkylene-N(R')(R"), C1-C6 alkyl, aryl, heteroaryl, each of which optionally contains one or more R sub optionally substituted with a group; R3 and R5 are each independently H, halo, OR 13 or C1-C6 alkylene-OR 13 , N(R')(R") or C1-C6 alkylene-N(R')(R"), C1-C6 alkyl, aryl, heteroaryl, each of which optionally contains one or more R sub or R3 and R5 together with the adjacent carbon atom and two sulfur atoms form a second ring of 6 to 8 atoms; R 13 is independently at each occurrence H, C-C alkyl, aryl, alkylcarbonyl, or arylcarbonyl; and R' and R" are each independently H or C1-C6 alkyl.

[0009] In one embodiment, the [cyclic disulfide moiety] is [ka] R2 has the structure: R2 can be an optionally substituted aryl, for example, an optionally substituted phenyl. In some embodiments, R2 is a mono-, di-, or tri-substituted phenyl. In some embodiments, R2 is a para-substituted phenyl. In some embodiments, R2 is an optionally substituted C1-6 alkyl. In some embodiments, R2 is a haloC1-6 alkyl. In some embodiments, R2 is C 1-6 It is alkyl.

[0010] In one embodiment, the [cyclic disulfide moiety] is [ka] R2 has the structure: R2 can be an optionally substituted aryl, for example, an optionally substituted phenyl. In some embodiments, R2 is a mono-, di-, or tri-substituted phenyl. In some embodiments, R2 is a para-substituted phenyl. In some embodiments, R2 is an optionally substituted C1-6 alkyl. In some embodiments, R2 is a haloC1-6 alkyl. In some embodiments, R2 is C 1-6 It is alkyl.

[0011] In some embodiments, the cyclic disulfide moiety has a structure selected from one of the following groups Ia), Ib), and II). Group Ia) includes the following structures: [ka] Group Ib) includes the following structures: [ka] Group II) includes the following structures: [ka]

[0012] In one embodiment, the phosphorus coupling group is: [ka] In these formulas, X1 and Z1 each independently represent H, OH, OM, OR 13 , S.H., S.M., S.R. 13 , C(O)H, S(O)H, or alkyl, each of which optionally is one or more R sub group, N(R')(R"), B(R 13 )3, BH3 - , optionally substituted with Se; or DQ, where D is independently at each occurrence absent, O, S, N(R'), alkylene, each of which optionally is substituted with one or more R sub group, and Q is independently at each occurrence a nucleoside or an oligonucleotide; X2 and Z2 are each independently N(R')(R"), OR 18 or DQ, where D is independently at each occurrence absent, O, S, N, N(R'), alkylene, each of which optionally is one or more R sub group, and Q is independently at each occurrence a nucleoside or an oligonucleotide; Y1 is S, O or N(R'); M is an organic or inorganic cation; and R 18 is H or optionally one or more R sub It is an alkyl substituted with a group.

[0013] In one embodiment, the phosphorus coupling group is [ka] In this formula, X1 and Z1 are each independently OH, OM, SH, SM, C(O)H, S(O)H, C1-C6 alkyl optionally substituted with one or more hydroxy or halo groups, or DQ; D is independently at each occurrence absent, O, S, NH, C1-C6 alkylene optionally substituted with one or more halo groups; and Y1 is S or O. In some embodiments, X1 is OH or SH; and Z1 is DQ.

[0014] In one embodiment, the phosphorus coupling group is [ka] In this formula, X2 is N(R')(R"); Z2 is X2, OR 18 or DQ;R 18 is H or C1-C6 alkyl substituted with cyano; and R' and R'' are each independently C1-C6 alkyl.

[0015] In one embodiment, the phosphorus coupling group is [ka] The variables R′, R″, and Q are as defined above in Formulas PI and P-II.

[0016] In some embodiments, the compound has one of the following structures: [ka] [ka] [ka]

[0017] In one embodiment, the phosphorus coupling group has the structure (PI), and the cyclic disulfide moiety -P(Y1)(X1)- is [ka] and X is O or S.

[0018] In some embodiments, the compound comprises one or more ligands connected to any one of R2, R3, R4, R5, R6, R7, R8 and R9 of the [cyclic disulfide moiety], optionally via one or more linkers.

[0019] In some embodiments, the ligand is selected from the group consisting of an antibody, a ligand-binding portion of a receptor, a ligand for a receptor, an aptamer, a carbohydrate-based ligand, a fatty acid, a lipoprotein, folate, thyrotropin, melanotropin, surfactant protein A, mucin, a glycosylated polyamino acid, transferrin, a bisphosphonic acid, a polyglutamic acid, a polyaspartic acid, a lipophilic moiety (e.g., a lipophilic moiety that enhances plasma protein binding), cholesterol, a steroid, a bile acid, vitamin B12, biotin, a fluorophore, and a peptide.

[0020] In some embodiments, at least one ligand is a carbohydrate-based ligand that targets liver tissue. In some embodiments, the carbohydrate-based ligand is selected from the group consisting of galactose, multivalent galactose, N-acetyl-galactosamine (GalNAc), multivalent GalNAc, mannose, multivalent mannose, lactose, multivalent lactose, N-acetyl-glucosamine (GlcNAc), multivalent GlcNAc, glucose, multivalent glucose, fucose, and multivalent fucose.

[0021] In certain embodiments, at least one ligand is a lipophilic moiety. In certain embodiments, the lipophilicity of the lipophilic moiety is determined by the log K owor the hydrophobicity of the compound, as measured by the unbound fraction in a plasma protein binding assay of the compound, is greater than 0.2.

[0022] In some embodiments, the lipophilic moiety is a saturated or unsaturated C4-C 30 The lipophilic moiety comprises a hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety may be a saturated or unsaturated C6-C 18 Contains a hydrocarbon chain.

[0023] In some embodiments, at least one ligand targets a receptor that mediates delivery to CNS tissue. In some embodiments, the ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, and LDL receptor ligand.

[0024] In some embodiments, at least one ligand targets a receptor that mediates delivery to an ocular tissue, hi some embodiments, the ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligands, and carbohydrate-based ligands.

[0025] Another aspect of the present invention is a compound of formula (I): [Cyclic disulfide moiety]-[phosphorus coupling group] (I) The present invention relates to an oligonucleotide (e.g., a single-stranded iRNA agent or a double-stranded iRNA agent) comprising the structure:

[0026] Another aspect of the present invention is a compound of formula (II): [Cyclic disulfide moiety]-P(Y)(X)- * (II) The present invention relates to an oligonucleotide (e.g., a single-stranded iRNA agent or a double-stranded iRNA agent) comprising the structure:

[0027] In both formulas (I) and (II), the [cyclic disulfide moiety] is [ka] or a salt thereof. R1 is O or S and is bonded to the P atom of formula (I) or the P atom of formula (II) of the [phosphorus coupling group]; R2, R4, R6, R7, R8 and R9 are each independently H, halo, OR 13 or alkylene-OR 13 , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R 14 )(R 15 )(R 16 ) or alkylene-C(R 14 )(R 15 )(R 16 ), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R sub optionally substituted with a group; R3 and R5 are each independently H, halo, OR 13 or alkylene-OR 13 , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R 14 )(R 15 )(R 16 ) or alkylene-C(R 14 )(R 15 )(R 16 ), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R sub or R3 and R5 together with the adjacent carbon atom and two sulfur atoms form a second ring; G is O, N(R'), S or C(R 14 )(R 15 ) and; n is an integer from 0 to 6; R 13is independently at each occurrence H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, each of which optionally contains one or more R sub optionally substituted with a group; R 14 , R 15 and R 16 are each independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, N(R')(R"); R′ and R″ are each independently H, alkyl, alkenyl, alkynyl, aryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, each of which optionally contains one or more R sub and R sub is independently at each occurrence halo, haloalkyl, alkyl, alkaryl, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, arylcarbonyl, acyloxy, cyano, or ureido; Here, when the [cyclic disulfide moiety] has the structure of formula (C-III), at least one [cyclic disulfide moiety] is connected at the 5' end of the nucleoside or oligonucleotide.

[0028] In formula (I), at least one of the phosphorus coupling groups comprises a nucleoside or oligonucleotide.

[0029] In formula (II), *represents a bond to an oligonucleotide, Y is absent, =O, or =S; X is OH, SH, or X', where X' is OR 13 or SR 13 is.

[0030] All of the formulae for [cyclic disulfide moiety] and [phosphorus coupling group] of Formula (CI) and Formula (C-II), all of the variables defined in these formulae, all of the ligands and all of the subgeneric and species structures associated with the compounds, and all of the above embodiments associated with [cyclic disulfide moiety] and [phosphorus coupling group] in the first aspect of the invention related to compounds are applicable to these aspects of the invention related to oligonucleotides.

[0031] In some embodiments, the cyclic disulfide moiety has a structure selected from one of the following groups Ia), Ib), and II). Group Ia) includes the following structures: [ka] Group Ib) includes the following structures: [ka] Group II) includes the following structures: [ka]

[0032] In some embodiments, the cyclic disulfide moiety has a structure selected from one of the structures from Group III). Group III) includes the following structures: [ka]

[0033] In one embodiment, the oligonucleotide [ka] or a salt thereof, wherein X is O or S.

[0034] In some embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(O)(SH)- * or a salt thereof.

[0035] In some embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(O)(OH)- * or a salt thereof.

[0036] In some embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(O)(OR 13 )- * or a salt thereof. 13 is as defined above.

[0037] In some embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(S)(OR 13 )- * or a salt thereof. 13 is as defined above.

[0038] In some embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(OR 13 )- * or a salt thereof. 13 is as defined above.

[0039] In one embodiment, the [cyclic disulfide moiety] is [ka] and having a structure selected from the group consisting of: *is -P(X)(Y)- * The bond of the group to the phosphorus atom is shown.

[0040] In some embodiments, the oligonucleotide comprises a structure selected from one of the following: [ka]

[0041] In one embodiment, the oligonucleotide [ka] X is O or S.

[0042] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at the 5' end of the oligonucleotide.

[0043] In some embodiments, the first nucleotide at the 5' end of the oligonucleotide has the structure: [ka] or a salt thereof. In these structures: * represents a subsequent bond to an optionally modified internucleotide linkage; Base is an optionally modified nucleobase; R S is [cyclic disulfide moiety]; and R is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH2-allyl, fluoro, O-N-methylacetamide (O-NMA), O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP), or ara-F. The variables X, X', and Y are as defined above in formula (II).

[0044] In some embodiments, the first nucleotide at the 5' end of the oligonucleotide has the structure: [ka] or a salt thereof. S , R 13 , R and Y are as defined above.

[0045] In some embodiments, the first nucleotide at the 5' end of the oligonucleotide has the structure: [ka] or a salt thereof. S and R is as defined above.

[0046] In some embodiments, Base in these structures is uridine. In some embodiments, R in these structures is methoxy. In some embodiments, R in these structures is hydrogen.

[0047] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at the 3' end of the oligonucleotide.

[0048] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at the 5' end of the oligonucleotide.

[0049] In certain embodiments, the oligonucleotide comprises at least one [cyclic disulfide moiety] at the 5' end of the oligonucleotide and at least one [cyclic disulfide moiety] at the 3' end of the oligonucleotide.

[0050] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at an internal position of the oligonucleotide.

[0051] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide.

[0052] In some embodiments, the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand.

[0053] In some embodiments, the sense strand and the antisense strand are each 15 to 30 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each 19 to 25 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each 21 to 23 nucleotides in length.

[0054] In some embodiments, the oligonucleotide comprises a single-stranded overhang on at least one end, e.g., a 3' and / or 5' overhang of 1 to 10 nucleotides in length, e.g., an overhang having a length of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. In some embodiments, the single-stranded overhang is optionally 1, 2, or 3 nucleotides in length on at least one end.

[0055] In some embodiments, the oligonucleotide may also have a blunt end at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. In some embodiments, the oligonucleotide comprises a 3' overhang at the 3' end of the antisense strand and optionally a blunt end at the 5' end of the antisense strand. In some embodiments, the oligonucleotide has a 5' overhang at the 5' end of the sense strand and optionally a blunt end at the 5' end of the antisense strand. In some embodiments, the oligonucleotide has two blunt ends at both ends of the double-stranded iRNA duplex.

[0056] In one embodiment, the sense strand of the oligonucleotide is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide-long single-stranded overhang at the 3' end.

[0057] In some embodiments, the sense strand comprises at least one cyclic disulfide moiety. In some embodiments, the antisense strand comprises at least one cyclic disulfide moiety. In some embodiments, both the sense strand and the antisense strand each comprise at least one cyclic disulfide moiety.

[0058] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at the 5'-end of the antisense strand and at least one targeting ligand at the 3'-end of the sense strand.

[0059] In certain embodiments, the sense strand further comprises at least one phosphorothioate linkage at the 3' end. In certain embodiments, the sense strand further comprises at least two phosphorothioate linkages at the 3' end.

[0060] In certain embodiments, the sense strand further comprises at least one phosphorothioate bond at the 5' end. In certain embodiments, the sense strand further comprises at least two phosphorothioate bond(s) at the 5' end.

[0061] In certain embodiments, the antisense strand further comprises at least one phosphorothioate bond at the 3' end. In certain embodiments, the antisense strand further comprises at least two phosphorothioate bond at the 3' end.

[0062] In certain embodiments, the oligonucleotide further comprises a phosphate or phosphate mimetic at the 5'-end of the antisense strand. In certain embodiments, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0063] In certain embodiments, the 5' end of the antisense strand does not contain a 5'-vinylphosphonate (VP).

[0064] In certain embodiments, the oligonucleotide further comprises at least one terminal, chiral phosphorus atom.

[0065] Site-specific chiral modifications of internucleotide linkages can occur at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the strand. This is referred to herein as "terminal" chiral modification. Terminal modifications can occur at the 3'- or 5'-end position in the terminal region, for example, at the terminal nucleotide of the strand or at a position within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Chiral modifications can occur in the sense strand, the antisense strand, or both the sense and antisense strands. Each chirally pure phosphorus atom can be in the Rp or Sp configuration, and combinations thereof. Further details about chiral modifications and chirally modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, entitled "Chirally Modified Double-Stranded RNA Agents," which is incorporated herein by reference in its entirety.

[0066] In some embodiments, the oligonucleotide further comprises a terminal chiral modification occurring at the first internucleotide bond at the 3' end of the antisense strand, the terminal chiral modification having the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide bond at the 5' end of the antisense strand, the terminal chiral modification having the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide bond at the 5' end of the sense strand, the terminal chiral modification having the linking phosphorus atom in the Rp or Sp configuration.

[0067] In one embodiment, the oligonucleotide further comprises a terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the sense strand, with the linking phosphorus atom in the Rp or Sp configuration.

[0068] In one embodiment, the oligonucleotide further comprises a terminal chiral modification occurring at the first, second, and third internucleotide bond at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide bond at the 5' end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide bond at the 5' end of the sense strand, with the linking phosphorus atom in the Rp or Sp configuration.

[0069] In one embodiment, the oligonucleotide further comprises a chiral modification at the first and second internucleotide bond at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a chiral modification at the third internucleotide bond at the 3' end of the antisense strand, with the linking phosphorus atom in the Rp configuration; a chiral modification at the first internucleotide bond at the 5' end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a chiral modification at the first internucleotide bond at the 5' end of the sense strand, with the linking phosphorus atom in the Rp or Sp configuration.

[0070] In one embodiment, the oligonucleotide further comprises a terminal chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first and second internucleotide linkages at the 5' end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5' end of the sense strand, with the linking phosphorus atom in the Rp or Sp configuration.

[0071] In certain embodiments, the oligonucleotide has at least two phosphorothioate internucleotide linkages in the first five nucleotides of the antisense strand (counting from the 5' end).

[0072] In some embodiments, the antisense strand comprises two blocks of 1, 2, or 3 phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0073] In some embodiments, the oligonucleotide comprises one or more targeting ligands connected, optionally via one or more linkers, to any one of R2, R3, R4, R5, R6, R7, R8, and R9 of the [cyclic disulfide moiety] of the compound.

[0074] In some embodiments, the targeting ligand is selected from the group consisting of an antibody, a ligand-binding portion of a receptor, a ligand for a receptor, an aptamer, a carbohydrate-based ligand, a fatty acid, a lipoprotein, folate, thyrotropin, melanotropin, surfactant protein A, mucin, a glycosylated polyamino acid, transferrin, a bisphosphonic acid, a polyglutamic acid, a polyaspartic acid, a lipophilic moiety that enhances plasma protein binding, cholesterol, a steroid, a bile acid, vitamin B12, biotin, a fluorophore, and a peptide.

[0075] In certain embodiments, at least one targeting ligand is a lipophilic moiety. In certain embodiments, the lipophilicity of the lipophilic moiety is determined by the log K ow or the hydrophobicity of the compound is greater than 0, as measured by the unbound fraction in a plasma protein binding assay of the compound, greater than 0.2. In certain embodiments, the lipophilic moiety is a saturated or unsaturated C4-C 30 The lipophilic moiety comprises a hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety may be a saturated or unsaturated C6-C 18 Contains a hydrocarbon chain.

[0076] In some embodiments, at least one targeting ligand targets a receptor that mediates delivery to a specific CNS tissue. In some embodiments, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, and LDL receptor ligand.

[0077] In some embodiments, at least one targeting ligand targets the receptor that mediates delivery to ocular tissue.In some embodiments, the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand and carbohydrate-based ligand.In some embodiments, the targeting ligand is RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or cyclo(-Arg-Gly-Asp-D-Phe-Cys).

[0078] In some embodiments, at least one targeting ligand targets liver tissue.In some embodiments, the targeting ligand is carbohydrate-based ligand.In some embodiments, the carbohydrate-based ligand is selected from the group consisting of galactose, multivalent galactose, N-acetyl-galactosamine (GalNAc), multivalent GalNAc, mannose, multivalent mannose, lactose, multivalent lactose, N-acetyl-glucosamine (GlcNAc), multivalent GlcNAc, glucose, multivalent glucose, fucose and multivalent fucose.In some embodiments, the targeting ligand is GalNAc conjugate.For example, GalNAc conjugate is: [ka] and one or more GalNAc derivatives linked via a bivalent or trivalent branched linker such as

[0079] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the antisense and sense strands of the oligonucleotide are modified. For example, when 50% of the oligonucleotide is modified, 50% of the total nucleotides present in the oligonucleotide contain a modification described herein.

[0080] In some embodiments, the antisense and sense strands of the oligonucleotide comprise at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or essentially 100% 2'-O-methyl modified nucleotides.

[0081] In certain embodiments, the oligonucleotide is a double-stranded dsRNA agent, and at least 50% of the nucleotides of the double-stranded dsRNA agent are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-deoxy, or 2'-fluoro.

[0082] In certain embodiments, the oligonucleotide is antisense and at least 50% of the nucleotides of the antisense are independently modified with LNA, CeNA, 2'-methoxyethyl, or 2'-deoxy.

[0083] In some embodiments, the sense strand and the antisense strand contain or do not contain 12 or less, 10 or less, 8 or less, 6 or less, 4 or less, 2 or less 2'-F modified nucleotides. In some embodiments, the oligonucleotide has or does not contain 12 or less, 10 or less, 8 or less, 6 or less, 4 or less, 2 or less 2'-F modifications in the sense strand. In some embodiments, the oligonucleotide has or does not contain 12 or less, 10 or less, 8 or less, 6 or less, 4 or less, 2 or less 2'-F modifications in the antisense strand. In some embodiments, the sense strand and the antisense strand contain 10 or less 2'-fluoro modified nucleotides.

[0084] In certain embodiments, the oligonucleotide comprises one or more 2'-O modifications selected from the group consisting of 2'-deoxy, 2'-O-methoxyalkyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0085] In certain embodiments, the oligonucleotide contains one or more 2'-F modifications anywhere in the sense or antisense strand.

[0086] In some embodiments, the oligonucleotide has less than 20%, less than 15%, less than 10%, less than 5% non-natural nucleotides, or is substantially free of non-natural nucleotides.Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modification (e.g., 2'-modified L-nucleoside, such as 2'-deoxy-L-nucleoside), BNA abasic sugar, abasic cyclic and open-chain alkyl.

[0087] In some embodiments, the oligonucleotide has more than 80%, more than 85%, more than 90%, more than 95%, or essentially 100% naturally occurring nucleotides. For purposes of these embodiments, naturally occurring nucleotides include those having 2'-OH, 2'-deoxy, and 2'-OMe.

[0088] In some embodiments, the antisense strand comprises at least one non-locked nucleic acid (UNA) or glycerol nucleic acid (GNA) modification, e.g., in the seed region of the antisense strand. In some embodiments, the seed region is positions 2-8 (or 5-7) of the 5' end of the antisense strand.

[0089] In some embodiments, the oligonucleotide has a sense strand and an antisense strand each having a length of 15-30 nucleotides; the first five nucleotides of the antisense strand contain at least two phosphorothioate internucleotide linkages (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); and wherein the oligonucleotide has less than 20%, less than 15%, less than 10%, less than 5% or is substantially free of non-natural nucleotides.

[0090] In certain embodiments, the oligonucleotides have sense and antisense strands each having a length of 15-30 nucleotides; the first five nucleotides of the antisense strand contain at least two phosphorothioate internucleotide linkages (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); and wherein the oligonucleotides have greater than 80%, greater than 85%, greater than 95%, or essentially 100% natural nucleotides, such as those having 2'-OH, 2'-deoxy, or 2'-OMe.

[0091] One embodiment of the present invention provides oligonucleotides comprising sense and antisense strands each independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides, counting from the 5' end of the antisense strand; and at least three, four, five, or six 2'-deoxy modifications in the sense and / or antisense strand; wherein the oligonucleotide has a double-stranded (duplex) region of 19 to 25 base pairs; and wherein the oligonucleotide comprises a ligand.

[0092] In some embodiments, the sense strand does not contain glycol nucleic acid (GNA).

[0093] It is understood that the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, in other words, the oligonucleotide is capable of inhibiting expression of the target gene.

[0094] In some embodiments, the oligonucleotide contains at least three 2'-deoxy modifications, the 2'-deoxy modifications being at positions 2 and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at position 11 of the sense strand, counting from the 5' end of the sense strand.

[0095] In some embodiments, the oligonucleotide contains at least five 2'-deoxy modifications, the 2'-deoxy modifications being at positions 2, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0096] In some embodiments, the oligonucleotide contains at least seven 2'-deoxy modifications, the 2'-deoxy modifications being at positions 2, 5, 7, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0097] In some embodiments, the antisense strand contains at least five 2'-deoxy modifications, counting from the 5' end of the antisense strand, at positions 2, 5, 7, 12, and 14. The antisense strand is 18-25 nucleotides in length or 18-23 nucleotides in length.

[0098] In certain embodiments, the oligonucleotide contains less than 20%, eg, less than 15%, less than 10%, or less than 5% non-natural nucleotides, or no non-natural nucleotides.

[0099] In some embodiments, the sense strand does not contain glycol nucleic acids (GNAs); and wherein the oligonucleotide contains less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides or contains all natural nucleotides.

[0100] In certain embodiments, at least one of the sense strand and the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more, 2'-deoxy modifications in the central region of the sense or antisense strand.

[0101] In some embodiments, the sense strand and / or antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more 2'-deoxy modifications in the central region of the sense strand and / or antisense strand.

[0102] In some embodiments, the sense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications in the central region of the sense strand, e.g., the sense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications within positions 7, 8, 9, 10, 11, 12, and 13, counting from the 5' end of the sense strand.

[0103] In some embodiments, the antisense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications in the central region of the antisense strand, e.g., the antisense strand is 18-30 nucleotides in length and comprises at least two 2'-deoxy modifications within positions 10, 11, 12, 13, 14, 15, and 16, counting from the 5' end of the antisense strand.

[0104] In one embodiment, the oligonucleotide comprises a sense strand and an antisense strand; wherein the sense strand is 17-30 nucleotides in length and comprises at least one 2'-deoxy modification in the central region of the sense strand; and wherein the antisense strand is independently 17-30 nucleotides in length and comprises at least two 2'-deoxy modifications in the central region of the antisense strand.

[0105] In one embodiment, the oligonucleotide comprises a sense strand and an antisense strand; wherein the sense strand is 17-30 nucleotides in length and comprises at least two 2'-deoxy modifications in the central region of the sense strand; and wherein the antisense strand is independently 17-30 nucleotides in length and comprises at least one 2'-deoxy modification in the central region of the antisense strand.

[0106] In certain embodiments, the sense strand includes at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the sense strand.

[0107] In certain embodiments, the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2'-deoxy modifications in the central region of the antisense strand.

[0108] In some embodiments, the oligonucleotide contains less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the oligonucleotide contains all natural nucleotides; and wherein the sense strand and / or antisense strand contains at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the sense strand and / or antisense strand.

[0109] In some embodiments, the oligonucleotide contains less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the oligonucleotide contains all natural nucleotides; and wherein the sense strand contains at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the sense strand.

[0110] In some embodiments, the oligonucleotide contains less than 20%, e.g., less than 15%, less than 10%, or less than 5% non-natural nucleotides, or the oligonucleotide contains all natural nucleotides; and wherein the antisense strand contains at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the antisense strand.

[0111] In certain embodiments, when the oligonucleotide contains less than 8 non-2'OMe nucleotides, the antisense strand contains at least one DNA. For example, in any of the embodiments of the present invention, when the oligonucleotide contains less than 8 non-2'OMe nucleotides, the antisense strand contains at least one DNA.

[0112] In some embodiments, when the antisense strand contains two deoxynucleotides, the nucleotides being at positions 2 and 14, counting from the 5' end of the antisense strand, the oligonucleotide contains eight or fewer (e.g., eight, seven, six, five, four, three, two, one, or zero) non-2'OMe nucleotides. For example, in any of the embodiments of the present invention, when the antisense strand contains two deoxynucleotides, the nucleotides being at positions 2 and 14, counting from the 5' end of the antisense strand, the oligonucleotide contains zero, one, two, three, four, five, six, seven, or eight non-2'-OMe nucleotides.

[0113] Another aspect of the present invention relates to a pharmaceutical composition comprising an oligonucleotide described herein and a pharmaceutically acceptable excipient.

[0114] All of the above embodiments relating to oligonucleotides in the above aspects of the invention relating to oligonucleotides are applicable to this aspect of the invention relating to pharmaceutical compositions.

[0115] In another aspect, the present invention further provides a method of delivering an oligonucleotide of the present invention to a specific target in a subject by subcutaneous or intravenous administration. The present invention further provides an oligonucleotide of the present invention for use in a method of delivering the agent to a specific target in a subject by subcutaneous or intravenous administration.

[0116] Another aspect of the invention relates to a method of reducing or inhibiting expression of a target gene in a subject, comprising administering to the subject an oligonucleotide as described above in an amount sufficient to inhibit expression of the target gene.

[0117] All of the above embodiments relating to oligonucleotides in the above aspects of the invention relating to oligonucleotides are applicable to this aspect of the invention relating to a method of reducing expression of a target gene in a subject.

[0118] Another aspect of the invention relates to a method of modifying an oligonucleotide, comprising contacting the oligonucleotide with the compound described above under conditions suitable for reaction of the compound with the oligonucleotide, wherein the oligonucleotide contains a free hydroxyl group.

[0119] In certain embodiments, the free hydroxyl group is part of the 5' terminal nucleotide. In certain embodiments, the free hydroxyl group is part of the 3' terminal nucleotide.

[0120] In certain embodiments, the oligonucleotide comprises a 5'-OH group. In certain embodiments, the oligonucleotide comprises a 3'-OH group.

[0121] In some embodiments, the conditions suitable for reacting the compound with the oligonucleotide include an acid catalyst. For example, the acid catalyst can be a substituted tetrazole. Suitable acid catalysts include 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, 5-nitrophenyl-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-benzylthio-1H-tetrazole, 5-methylthio-1H-tetrazole, 1-hydroxybenzotriazole, 1-hydroxy-6-trifluoromethylbenzotriazole, 4-nitro-1-hydroxy-6-trifluoromethylbenzotriazole, 1-hydroxy ... Triazole, pyridinium chloride, pyridinium bromide, pyridinium 4-methylbenzenesulfonate, 2,6-di(tert-butyl)pyridinium chloride, pyridinium trifluoroacetate, N-(phenyl)imidazolium triflate (N-PhIMT), N-(phenyl)-imidazolium perchlorate (N-PhIMP), N-(methyl)benzimidalium triflate (NMeBIT), N-(p-acetylphenyl)imidalium triflate (N-AcPhIMT), N-(phenyl)imidazolium tetrachloride tetrafluoroborate (N-PhIMTFB), imidazolium perchlorate (IMP), 4-(phenyl)-imidalium triflate (4-PhIMT), benzimidazolium tetrafluoroborate (BITFB), imidazolium tetrafluoroborate (IMTFB), imidalium triflate (IMT), benzimidalium triflate (BIT), 2-(phenyl)imidalium triflate (2-PhIMT), N-(methyl)imidalium triflate (N-MeIMT), 4-(methyl)imidalium Imidalium triflate (4-MeIMT), saccharin-1-methylimidazole, N-(cyanomethyl)pyrrolidinium triflate, trichloroacetic acid (TCA), trifluoroacetic acid (TFA), dichloroacetic acid (DCA) and 2,4-dinitrobenzoic acid (2,4-DNBA), iron chloride (FeCl3), aluminum chloride (AlCl3), boron trifluoroetherate (BF3-OEt2), zirconium(IV) chloride (ZrCl4) and bismuth(III) chloride (BiCl3), trimethylchlorosilane, 2,These include, but are not limited to, 4-dinitrophenol, 1-methyl-5-mercaptotetrazole, and 1-phenyl-5-mercaptotetrazole.

[0122] All of the above embodiments relating to compounds and oligonucleotides in the above aspects of the invention are applicable to this aspect of the invention relating to methods of modifying oligonucleotides.

[0123] Another aspect of the invention is a method for making a modified oligonucleotide, comprising the steps of: Formula (A): [ka] [During the ceremony, R S is [cyclic disulfide moiety]; X' is -OR 13 or -SR 13 where R 13 is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, each of which optionally contains one or more R sub may be substituted with a group. or a salt thereof, with a first oligonucleotide having formula (B): [ka] wherein Y is O or S; and X is —OH, —SH, or X′. or a salt thereof. The present invention relates to a method for generating a variable group Base, R S , X, X' and Y are as defined above.

[0124] In some embodiments, the first nucleotide at the 5' end of the first oligonucleotide comprises a group of Formula (A) and the first nucleotide at the 5' end of the modified oligonucleotide comprises a group of Formula (B). In some embodiments, the last nucleotide at the 3' end of the first oligonucleotide comprises a group of Formula (A) and the last nucleotide at the 3' end of the modified oligonucleotide comprises a group of Formula (B).

[0125] In some embodiments, the first nucleotide at the 5' end of the first oligonucleotide has Formula (C): [ka] or a salt thereof, wherein * represents a subsequent bond to an optionally modified internucleotide linkage; Base is an optionally modified nucleobase; and R is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH2-allyl, fluoro, O-N-methylacetamide (O-NMA), O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP), or ara-F. S and X' is as defined above.

[0126] In some embodiments, the first nucleotide at the 5' end of the modified oligonucleotide has Formula (D): [ka] The variable groups Base, R, and R S , X and Y are as defined above.

[0127] In some embodiments, the first nucleotide at the 5' end of the modified oligonucleotide has Formula (E) or (F): [ka] or a salt thereof, wherein **represents the bond to the subsequent nucleotide. Variables Base, R, R S , X and Y are as defined above.

[0128] In some embodiments, the conditions suitable for forming modified oligonucleotide include an oxidizing agent selected from the group consisting of iodine; sulfur; peroxide; peracid; phenylacetyl disulfide; 3H-1,2-benzodithiol-3-one 1,1-dioxide; dixanthogen; 5-ethoxy-3H-1,2,4-dithiazol-3-one; 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazol-5-thione (DDTT); dimethyl sulfoxide; and N-bromosuccinimide.For example, the oxidizing agent can be peracid (for example, m-chloroperbenzoic acid) or peroxide (for example, tert-butyl hydroperoxide or trimethylsilyl peroxide).

[0129] All of the above embodiments relating to compounds and oligonucleotides in the above aspects of the invention are applicable to this aspect of the invention relating to methods of making modified oligonucleotides. [Brief explanation of the drawings]

[0130] [Figure 1] Figure 1 is a graph showing the in vitro activity of F12 siRNA containing a modified phosphate prodrug at the 5' end in primary mouse hepatocytes analyzed after transfection with 0.1 nm, 1 nm, 10 nm, and 100 nm concentrations of RNAiMAX and 24 hours post-transfection. The percentage of remaining F12 message was determined by qPCR and plotted relative to the control.

[0131] [Figure 2]Figure 2 is a graph showing the in vitro activity of F12 siRNA duplexes containing a modified phosphate prodrug at the 5' end in primary mouse hepatocytes analyzed after incubation at 0.1 nm, 1 nm, 10 nm, and 100 nm concentrations and after 48 hours of incubation. The percentage of remaining F12 message was determined by qPCR and plotted relative to the control.

[0132] [Figure 3] Figure 3 is a graph showing the in vitro activity of F12 siRNA duplexes containing a modified phosphate prodrug at the 5' end in primary mouse hepatocytes analyzed after transfection with 0.1 nm, 1 nm, and 10 nm concentrations of RNAiMAX and 24 hours post-transfection. The percentage of remaining F12 message was determined by qPCR and plotted relative to the control.

[0133] [Figure 4] Figures 4A-J show representative LCMS spectra of oligonucleotides tested in the DTT reduction assay.

[0134] [Figure 5] FIG. 5 is a graph showing relative circulating mF12 protein by ELISA in mice following subcutaneous administration of a single 0.3 mg / kg dose of F12 siRNA duplex containing a modified phosphate prodrug at the 5′ end compared to a PBS control.

[0135] [Figure 6] Figure 6 is a graph showing relative circulating mF12 protein by ELISA in mice after subcutaneous administration of a single dose of 0.1 mg / kg or 0.3 mg / kg of F12 siRNA duplex containing a modified phosphate prodrug at the 5' end compared to a PBS control.

[0136] [Figure 7] FIG. 7 shows a possible in vivo cytoplasmic unmasking mechanism of a 5′ cyclic disulfide-modified phosphate prodrug to expose the 5′-phosphate.

[0137] [Figure 8] Figure 8 is a graph showing the relative SOD1 mRNA remaining in the thoracic spinal cord, hippocampus, frontal cortex, striatum, and heart of rats, as determined by qPCR, 14 days after intrathecal (IT) administration of a single 0.1 mg dose of SOD1 siRNA duplex containing a modified phosphate prodrug at the 5' end.

[0138] [Figure 9] Figure 9 is a graph showing the relative SOD1 mRNA remaining in the thoracic spinal cord, cerebellum, frontal cortex, striatum, and heart of rats, as determined by qPCR, 84 days after intrathecal (IT) administration of a single 0.3 mg dose of SOD1 siRNA duplex containing a modified phosphate prodrug at the 5' end.

[0139] [Figure 10] Figure 10 is a graph showing the relative SOD1 mRNA remaining in the thoracic spinal cord, hippocampus, frontal cortex, striatum, and heart of rats, as determined by qPCR, 14 days after intrathecal (IT) administration of a single 0.9 mg dose of SOD1 siRNA duplex containing a modified phosphate prodrug at the 5' end.

[0140] [Figure 11] Figure 11 is a graph showing the relative SOD1 mRNA remaining in the thoracic spinal cord, cerebellum, frontal cortex, striatum, and heart of rats, as determined by qPCR, 84 days after intrathecal (IT) administration of a single 0.9 mg dose of SOD1 siRNA duplex containing a modified phosphate prodrug at the 5' end.

[0141] [Figure 12] Figure 12 is a graph showing the relative remaining SOD1 mRNA by qPCR in the thoracic spinal cord, hippocampus, frontal cortex, striatum, and heart of rats 14 days after intrathecal administration of a single 0.9 mg dose of SOD1 siRNA duplex containing a modified phosphate prodrug at the 5' end.

[0142] [Figure 13]Figure 13 is a graph showing the relative remaining SOD1 mRNA by qPCR in the thoracic spinal cord, hippocampus, frontal cortex, striatum, and heart of rats 14 days after intrathecal administration of a single dose of 0.3 mg or 0.9 mg of SOD1 siRNA duplex containing a modified phosphate prodrug at the 5' end.

[0143] [Figure 14] Figure 14 is a graph showing the relative remaining SOD1 mRNA by qPCR in the right hemisphere of mice 7 days after intracranial administration of a single 100 μg dose of SOD1 siRNA duplex containing a modified phosphate prodrug at the 5' end. DETAILED DESCRIPTION OF THE INVENTION

[0144] Detailed Description The present inventors have discovered a novel category of cyclic disulfide moieties that can be introduced into phosphate groups of oligonucleotides (e.g., single-stranded and double-stranded iRNA agents) to transiently mask the phosphate group and can be cleaved in vivo via cellular activation. Cellular activation is via a glutathione- or dithiothreitol-mediated reduction / bioconvention mechanism to release the phosphate group in active anionic form from the masking group. The present inventors have discovered that cyclic disulfide moieties can be introduced into the sense strand, antisense strand, or both the sense and antisense strands at the 5'-end, 3'-end, and / or internal positions of the strands. Introduction of a cyclic disulfide moiety-modified phosphate prodrug at the 5'-end of the antisense strand provides particularly good results.

[0145] Modified Phosphate Prodrug Compounds One aspect of the present invention relates to modified phosphate prodrug compounds having formula (I): [Cyclic disulfide moiety]-[phosphorus coupling group] (I) Includes the structure of

[0146] [Cyclic disulfide moiety] is: [ka] It has the following structure.

[0147] In formulae (CI), (C-II) and (C-III), R1 is O or S and is attached to the P atom of the [phosphorus coupling group]; R2, R4, R6, R7, R8 and R9 are each independently H, halo, OR 13 or alkylene-OR 13 , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R 14 )(R 15 )(R 16 ) or alkylene-C(R 14 )(R 15 )(R 16 ), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R sub optionally substituted with a group; R3 and R5 are each independently H, halo, OR 13 or alkylene-OR 13 , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R 14 )(R 15 )(R 16 ) or alkylene-C(R 14 )(R 15 )(R 16 ), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R sub or R3 and R5 together with the adjacent carbon atom and two sulfur atoms form a second ring; G is O, N(R'), S or C(R 14 )(R 15 ) and; n is an integer from 0 to 6; R 13is independently at each occurrence H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, each of which optionally contains one or more R sub optionally substituted with a group; R 14 , R 15 and R 16 are each independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, N(R')(R"); R′ and R″ are each independently H, alkyl, alkenyl, alkynyl, aryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, each of which optionally contains one or more R sub and R sub is independently at each occurrence halo, haloalkyl, alkyl, alkaryl, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, arylcarbonyl, acyloxy, cyano, or ureido.

[0148] In one embodiment, in the [cyclic disulfide moiety]: R1 is O; G is CH2; n is 0 or 1; R2, R4, R6, R7, R8 and R9 are each independently H, halo, OR 13 or C1-C6 alkylene-OR 13, N(R')(R") or C1-C6 alkylene-N(R')(R"), C1-C6 alkyl, aryl, heteroaryl, each of which optionally contains one or more R sub optionally substituted with a group; R3 and R5 are each independently H, halo, OR 13 or C1-C6 alkylene-OR 13 , N(R')(R") or C1-C6 alkylene-N(R')(R"), C1-C6 alkyl, aryl, heteroaryl, each of which optionally contains one or more R sub or R3 and R5 together with the adjacent carbon atom and two sulfur atoms form a second ring of 6 to 8 atoms; R 13 is independently at each occurrence H, C-C alkyl, aryl, alkylcarbonyl, or arylcarbonyl; and R' and R" are each independently H or C1-C6 alkyl.

[0149] [Phosphorus coupling group] [ka] It may have the structure:

[0150] In formulas (PI) and (P-II), X1 and Z1 each independently represent H, OH, OM, OR 13 , S.H., S.M., S.R. 13 , C(O)H, S(O)H, or alkyl, each of which optionally is one or more R sub group, N(R')(R"), B(R 13 )3, BH3 - , optionally substituted with Se; or DQ, where D is independently at each occurrence absent, O, S, N(R'), alkylene, each of which optionally is substituted with one or more R sub group, and Q is independently at each occurrence a nucleoside or an oligonucleotide; X2 and Z2 are each independently N(R')(R"), OR 18or DQ, where D is independently at each occurrence absent, O, S, N, N(R'), alkylene, each of which optionally is one or more R sub group, and Q is independently at each occurrence a nucleoside or an oligonucleotide; Y1 is S, O or N(R'); M is an organic or inorganic cation; and R 18 is H or optionally one or more R sub It is an alkyl substituted with a group.

[0151] In one embodiment, the phosphorus coupling group is [ka] In this formula, X1 and Z1 are each independently OH, OM, SH, SM, C(O)H, S(O)H, C1-C6 alkyl optionally substituted with one or more hydroxy or halo groups, or DQ; D is independently at each occurrence absent, O, S, NH, C1-C6 alkylene optionally substituted with one or more halo groups; and Y1 is S or O. In some embodiments, X1 is OH or SH; and Z1 is DQ.

[0152] In one embodiment, the phosphorus coupling group is [ka] wherein X1 is OH or SH.

[0153] In one embodiment, the phosphorus coupling group is [ka] In this formula, X2 is N(R')(R"); Z2 is X2, OR 18 or DQ;R 18is H or C1-C6 alkyl substituted with cyano; and R' and R'' are each independently C1-C6 alkyl (e.g., iso-propyl).

[0154] In one embodiment, the phosphorus coupling group is [ka] The variables R', R" and Q are as defined above in formulas PI and P-II. In some embodiments, R' and R" are each iso-propyl.

[0155] In some embodiments, the phosphorus coupling group has the structure -P(Z)(X), where: X is -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH2CH(CH3)2, -OCH2CH2CN, -OCH2CH2Si(CH3)3, -OCH2CH2Si(CH2CH3)3, -OC(H)=CH2, -OCH2C(H)=CH2 [ka] selected from the group consisting of: Z is [ka] or X and Z are bonded together with the phosphorus atom to which they are attached. [ka] and forming a cyclic structure selected from the group consisting of:

[0156] In one embodiment, the phosphorus coupling group is [ka] It has the following structure.

[0157] [Cyclic disulfide moiety] has the structure [ka] may have:

[0158] Examples of cyclic disulfide moieties of five-membered cyclic compounds of formula (CI) are [ka] Includes.

[0159] In some embodiments, the compound [ka] wherein R2, R3, R4, and R5 are each independently H, alkyl (e.g., CH3), a heterocycle, CH2R 15 , aryl (e.g., phenyl), heteroaryl, CHFR 15 , CF2R 15 CF3; and may be in any stereoisomeric configuration; and R 15 is alkyl, heterocycle, aryl, OH, O-alkyl, NH2, NH(alkyl), N(alkyl)2, CF2R 15 or CF3; and can be in any stereoisomeric configuration.

[0160] Examples of compounds of formula (I) having a five-membered cyclic disulfide moiety are shown in Table 1. [Table 1] [Table 2]

[0161] In one embodiment, the [cyclic disulfide moiety] has the structure [ka] wherein R3 and R5 together with the adjacent carbon atom and two sulfur atoms form a second ring. In some embodiments, the second ring has 6 to 8 atoms.

[0162] Examples of the [cyclic disulfide moiety] of the bicyclic compound of formula (CI) are [ka] Includes.

[0163] In some embodiments, the compound [ka] where R3 and R5 together with adjacent carbon atoms and two sulfur atoms form a second ring (eg, having 6 to 8 atoms).

[0164] Examples of compounds of formula (I) having a bicyclic disulfide moiety are shown in Table 2. [Table 3]

[0165] The cyclic disulfide moiety also has the structure [ka] Examples of the cyclic disulfide moiety of the cyclic compound of formula (C-II) include: [ka] Includes.

[0166] In some embodiments, the compound [ka] In these formulas, n is 1, 2, 3, 4, 5, or 6; G is O, NR 15, S, or any other heteroatom; R2, R3, R4, R5, and R6 are each independently H, alkyl (e.g., CH3), a heterocycle, CH2R 15 , aryl (e.g., phenyl), heteroaryl, CHFR 15 , CF2R 15 CF3; and may be in any stereoisomeric configuration; and R 15 is alkyl, heterocycle, aryl, OH, O-alkyl, NH2, NH(alkyl), N(alkyl)2, CF2R 15 or CF3; and can be in any stereoisomeric configuration.

[0167] Examples of compounds of formula (I) having large (7 or more members) cyclic disulfide moieties are shown in Table 3. [Table 4]

[0168] [Cyclic disulfide moiety] has the structure [ka] Examples of the [cyclic disulfide moiety] of the 6-membered cyclic compound of formula (C-III) are: [ka] Includes.

[0169] In some embodiments, the compound [ka] wherein R2, R3, R4, R5, and R6 are each independently H, alkyl (e.g., CH3), a heterocycle, CH2R 15 , aryl (e.g., phenyl), heteroaryl, CHFR 15 , CF2R 15 CF3; and may be in any stereoisomeric configuration; and R 15is alkyl, heterocycle, aryl, OH, O-alkyl, NH2, NH(alkyl), N(alkyl)2, CF2R 15 or CF3; and can be in any stereoisomeric configuration.

[0170] Examples of compounds of formula (I) having a six-membered cyclic disulfide moiety are shown in Table 4. [Table 5]

[0171] Certain terms, including, for example, methyl (Me), benzoyl (Bz), phenyl (Ph), and pivaloyl (Piv), are abbreviated in chemical structures throughout this specification as is well known to those of skill in the art.

[0172] The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine, or iodine.

[0173] As used herein, the term "aliphatic" or "aliphatic group" refers to a straight or branched, substituted or unsubstituted hydrocarbon chain that is saturated or contains one or more units of unsaturation, or a monocyclic hydrocarbon, bicyclic, or polycyclic hydrocarbon that is saturated or contains one or more units of unsaturation, but is not aromatic, having one point of attachment to the rest of the molecule. In some embodiments, an aliphatic group contains 1 to 50 aliphatic carbon atoms, e.g., 1 to 10 aliphatic carbon atoms, 1 to 6 aliphatic carbon atoms, 1 to 5 aliphatic carbon atoms, 1 to 4 aliphatic carbon atoms, 1 to 3 aliphatic carbon atoms, or 1 to 2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" refers to a monocyclic or bicyclic C3-C6 ring that is saturated or contains one or more units of unsaturation, but is not aromatic, having one point of attachment to the rest of the molecule. 10 "Aliphatic groups" refers to a hydrocarbon (e.g., a monocyclic C3-C6 hydrocarbon). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0174] The term "alkyl" refers to a hydrocarbon chain that may be straight or branched, containing the indicated number of carbon atoms. For example, C1-C 12 Alkyl indicates that the group may contain 1 to 12 (inclusive) carbon atoms in it. Unless otherwise specified, "alkyl" generally refers to C1-C 24 Alkyl (e.g., C1-C 12 The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens are replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally interrupted by O, N, or S. The term "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom is replaced by an aryl group. Examples of "aralkyl" include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

[0175] The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2 to 8 carbon atoms and characterized by having one or more double bonds. Unless otherwise specified, "alkenyl" generally refers to C2-C8 alkenyl (e.g., C2-C6 alkenyl, C2-C4 alkenyl, or C2-C3 alkenyl). Typical alkenyl examples include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenyl groups. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 8 carbon atoms and characterized by having one or more triple bonds. Unless otherwise specified, "alkynyl" generally refers to C2-C8 alkynyl (e.g., C2-C6 alkynyl, C2-C4 alkynyl, or C2-C3 alkynyl). Some examples of typical alkynyl groups are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. 2 and sp 3 The carbons can optionally serve as the points of attachment for the alkenyl and alkynyl groups, respectively.

[0176] The term "alkoxy" refers to an -O-alkyl group. The term "alkylene" refers to a divalent alkyl (i.e., -R-). The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl group.

[0177] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH) n -, where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. A substituted alkylene chain refers to a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include:

[0178] The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain refers to a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include:

[0179] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system, in which 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term "aryl" may be used interchangeably with the term "aryl ring." Aryl groups include phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused with one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl. The term "arylalkyl" or "aralkyl" refers to an alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with an aryl.

[0180] As used herein, the term "cycloalkyl" or "cyclyl" includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons and, e.g., 3 to 6 carbons, where the cycloalkyl group may be further optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0181] The terms "heteroaryl" or "heteroar-" refer to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms if monocyclic, 1 to 6 heteroatoms if bicyclic, or 1 to 9 heteroatoms if tricyclic, the heteroatoms being selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), where 0, 1, 2, 3, or 4 atoms of each ring may be substituted. The terms also include groups in which the heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of heteroaryl groups are pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, Examples of heteroaryl alkyl include benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. The term "heteroarylalkyl" or "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with a heteroaryl.

[0182] The terms "heterocyclyl," "heterocycle," "heterocyclic radical," or "heterocycle" refer to a non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms if monocyclic, 1 to 6 heteroatoms if bicyclic, or 1 to 9 heteroatoms if tricyclic, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), where 0, 1, 2, or 3 atoms in each ring may be substituted by a substituent. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). Examples of heterocyclyl groups include triazolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, etc. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions independently are optionally substituted.

[0183] The term "oxo" refers to an oxygen atom which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0184] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

[0185] The term "substituted" refers to the replacement of one or more hydrogen radicals in a structure with the radical of a specified substituent, including, but not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent may be further substituted.

[0186] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O- or -S(C(R * 2)) 2-3 S-(where R * is independently at each occurrence hydrogen, optionally substituted C as defined below 1-6 A suitable divalent substituent attached to a vicinal substitutable carbon of an "optionally substituted" group is -O(CR * 2) 2-3 O-, where R *is independently at each occurrence hydrogen, optionally substituted C as defined below 1-6 It is selected from aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0187] Oligonucleotide Prodrugs Another aspect of the present invention is a compound of formula (I): [Cyclic disulfide moiety]-[phosphorus coupling group] (I) In formula (I), at least one [phosphorus coupling group] comprises a nucleoside or oligonucleotide.

[0188] All of the above embodiments relating to all of the formulae for [Cyclic Disulfide Moiety], all of the formulae for [Phosphorus Coupling Group], all of the variables defined in these formulae, and all of the subgenera and species structures relating to the compounds of the first aspect of the invention, [Cyclic Disulfide Moiety] and [Phosphorus Coupling Group] (or modified phosphate prodrug compounds) are applicable to these aspects of the invention relating to oligonucleotides.

[0189] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at the 5' end of the oligonucleotide.

[0190] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at the 3' end of the oligonucleotide.

[0191] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at an internal position of the oligonucleotide.

[0192] In certain embodiments, when the cyclic disulfide moiety has the structure of formula (C-III), at least one cyclic disulfide moiety is attached at the 5' end of the nucleoside or oligonucleotide.

[0193] Further structures for modified phosphate prodrug compounds include those disclosed in WO2014 / 088920, published June 12, 2014, the contents of which are incorporated herein by reference in their entirety. In particular, these modified phosphate prodrug compounds are incorporated into oligonucleotides at the 5' end.

[0194] In certain embodiments, the oligonucleotide is a single-stranded oligonucleotide, such as a single-stranded iRNA agent (e.g., a single-stranded iRNA).

[0195] In certain embodiments, the oligonucleotide is a double-stranded oligonucleotide, such as a double-stranded iRNA agent comprising a sense strand and an antisense strand (e.g., a double-stranded iRNA).

[0196] In some embodiments, the sense strand comprises at least one cyclic disulfide moiety. In some embodiments, the antisense strand comprises at least one cyclic disulfide moiety. In some embodiments, both the sense strand and the antisense strand each comprise at least one cyclic disulfide moiety.

[0197] Introduction of a phosphate group into the [cyclic disulfide moiety] as a temporary protecting group into the sense strand, the antisense strand, or both the sense and antisense strands is illustrated in Schemes 10 to 15 of Example 9 below.

[0198] Oligonucleotide definition and design Unless otherwise defined, the nomenclature and procedures and techniques utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Some such techniques and procedures can be found, for example, in "Carbohydrate Modifications in Antisense Research," Edited by Sangvi and Cook, American Chemical Society, Washington, DC, 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and "Antisense Drug Technology, Principles, Strategies, and Applications," Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A Laboratory Manual," 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. Where permitted, all patents, applications, published applications and other publications and other data referenced throughout this specification are incorporated herein by reference in their entirety.

[0199] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be regulated by siRNA compounds.Target nucleic acids include, but are not limited to, RNA (including but not limited to pre-mRNA and mRNA or a part thereof) translated from DNA encoding target protein, and also cDNA derived from such RNA and miRNA.For example, target nucleic acids can be cellular genes (or mRNA transcribed from genes) whose expression is associated with a particular disorder or disease state.In some embodiments, target nucleic acids can be nucleic acid molecules from infectious agents.

[0200] As used herein, the term "iRNA" refers to an agent that mediates the targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Agents effective in inducing RNA interference are referred to herein as siRNAs, RNAi agents, or iRNA agents. Thus, these terms may be used interchangeably herein. As used herein, the term iRNA includes microRNAs and pre-microRNAs. Furthermore, as used herein, the term "compound" of the invention also refers to an iRNA agent and may be used interchangeably with iRNA agent.

[0201] An iRNA agent should contain a region of sufficient homology with a target gene and be of sufficient length, in terms of nucleotides, so that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. (For ease of exposition, the terms nucleotide or ribonucleotide may be used herein to refer to one or more monomeric subunits of an iRNA agent. It should be understood that the use of the terms "ribonucleotide" or "nucleotide" herein also refers, in the case of a modified RNA or nucleotide surrogate, to the modified nucleotide or surrogate replacement moiety at one or more positions.) Thus, an iRNA agent is or contains a region that is at least partially, and in some embodiments, completely, complementary to a target RNA. While perfect complementarity between an iRNA agent and a target is not required, the correspondence must be sufficient so that the iRNA agent or its cleavage product can direct sequence-specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. The degree of complementarity or homology with the target strand is most important in the antisense strand. While perfect complementarity is often desired, particularly in the antisense strand, certain embodiments can contain one or more, or, for example, 6, 5, 4, 3, 2, or fewer, mismatches (relative to the target RNA), particularly in the antisense strand. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-stranded character of the molecule.

[0202] iRNA agents include molecules that are long enough to initiate an interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)); and molecules that are short enough not to initiate an interferon response (which can also be cleaved by Dicer and / or enter RISC), e.g., molecules of a size that allows them to enter RISC, e.g., molecules that mimic Dicer cleavage products. Molecules that are short enough not to initiate an interferon response are referred to herein as siRNA agents or short iRNA agents. As used herein, "siRNA agent or short iRNA agent" refers to an iRNA agent, e.g., a double-stranded RNA agent or a single-stranded agent, that is short enough not to initiate a deleterious interferon response in human cells, e.g., having a duplex region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent or its cleavage product can downregulate a target gene, for example, by inducing RNAi against the target RNA, where the target can include an endogenous or pathogen target RNA.

[0203] As used herein, a "single-stranded iRNA agent" is an iRNA agent that consists of a single molecule. It may contain a duplex region formed by intrastrand pairing, e.g., it may be or include a hairpin or panhandle structure. A single-stranded iRNA agent may be antisense to a target molecule. A single-stranded iRNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of a target mRNA. A single-stranded iRNA agent is at least 14, and in other embodiments, at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.

[0204] A loop refers to a region of an iRNA strand that is unpaired to the opposing nucleotide in the duplex when the iRNA strand is base-paired to another strand or to another strand of the same strand.

[0205] Hairpin iRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs or more. The duplex region is 200, 100, or 50 or less in length. In certain embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length. The hairpin can have a single-stranded overhang or terminal unpaired region, in some embodiments on the 3' and in some embodiments on the antisense side of the hairpin. In certain embodiments, the overhang is 2-3 nucleotides in length.

[0206] As used herein, a "double-stranded (ds) iRNA agent" refers to an iRNA agent that contains more than one, and in some cases two, strands where interstrand hybridization can form a region of duplex structure.

[0207] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0208] As used herein, "gene silencing" by an RNA interference molecule refers to a reduction in the mRNA level in a cell of a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% up to 100%, inclusive, and any integer between 5% and 100%, of the mRNA level found in a cell in the absence of an miRNA or RNA interference molecule. In a preferred embodiment, the mRNA level is reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, inclusive, and any integer between 5% and 100%.

[0209] As used herein, the term "modulation of gene expression" means that the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits is up-regulated or down-regulated such that the expression, level, or activity is greater or less than that seen in the absence of a modulator. For example, the term "modulation" can mean "inhibition," although the use of the word "modulation" is not limited to this definition.

[0210] As used herein, gene expression modulation occurs when the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more from that observed in the absence of an siRNA, e.g., an RNAi agent. The percent and / or fold difference can be calculated relative to a control or non-control, for example,

number

number

[0211] As used herein, the terms "inhibition," "downregulation," or "reduction" in relation to gene expression means that the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced to a level below that observed in the absence of a modulator. Gene expression is downregulated when the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced by at least 10%, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably 100% lower (i.e., no gene expression) relative to a corresponding unmodulated control.

[0212] As used herein, the term "increase" or "upregulation," in relation to gene expression, refers to an increase in the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, above that observed in the absence of a modulator. Gene expression is upregulated when the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased by at least 10%, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or greater, relative to a corresponding unmodulated control.

[0213] As used herein, the term "increased" or "increase" generally refers to an increase by a statistically significant amount; for the avoidance of doubt, "increased" means at least a 10% increase compared to a reference level, for example, at least about a 20% increase compared to a reference level, or at least about a 30% increase, or at least about a 40% increase, or at least about a 50% increase, or at least about a 60% increase, or at least about a 70% increase, or at least about a 80% increase, or at least about a 90% increase, or up to a 100% increase (inclusive) compared to a reference level, or any increase between 10-100%, or at least about a 2-fold increase, or at least about a 3-fold increase, or at least about a 4-fold increase, or at least about a 5-fold increase, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0214] As used herein, the term "decreased" or "reduction" generally refers to a statistically significant reduction. However, for the avoidance of doubt, "decreased" refers to a reduction of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to (inclusive) a reduction of at least about 100% compared to a reference level (i.e., a level that is absent compared to a reference sample), or any reduction between 10 and 100%.

[0215] A double-stranded iRNA comprises two oligonucleotide strands sufficiently complementary to hybridize to form a duplex structure. Typically, the duplex structure is 15-30, more typically 18-25, even more typically 19-24, and most typically 19-21 base pairs in length. In some embodiments, long double-stranded iRNAs of 25-30 base pairs in length are preferred. In some embodiments, short double-stranded iRNAs of 10-15 base pairs in length are preferred. In other embodiments, the double-stranded iRNA is at least 21 nucleotides in length.

[0216] In some embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity that is complementary to at least a portion of a target sequence, and the duplex region is 14-30 nucleotides in length. Similarly, the region of complementarity to the target sequence is 14-30, more typically 18-25, even more typically 19-24, and most typically 19-21 nucleotides in length.

[0217] The term "antisense strand" as used herein refers to an oligonucleotide strand that is substantially or 100% complementary to the target sequence of interest. The term "antisense strand" includes both oligonucleotide strands formed from two separate strands, as well as the antisense region of a single-molecule oligonucleotide strand that can form a hairpin or dumbbell-shaped structure. The terms "antisense strand" and "guide strand" are used interchangeably herein.

[0218] The term "sense strand" refers to an oligonucleotide strand that has the same nucleoside sequence, in whole or in part, as a target sequence, such as a sequence of messenger RNA or DNA. The terms "sense strand" and "passenger strand" are used interchangeably herein.

[0219] " Specifically hybridizable " and " complementary " mean that a nucleic acid can hydrogen bond with other nucleic acid sequence by classical Watson or other non-classical type.With respect to the nucleic acid molecule of the present invention, the binding free energy between nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of nucleic acid, such as RNAi activity, to proceed.The determination of the binding free energy of nucleic acid molecule is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100% complementarity means that all contiguous residues in a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than perfect complementarity refers to information in which some, but not all, nucleoside units of two strands can hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected to be non-complementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, or conditions under which the assay is performed in the case of assays. Non-target sequences typically differ by at least 5 nucleotides.

[0220] In some embodiments, the double-stranded region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0221] In some embodiments, the antisense strand is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides in length.

[0222] In some embodiments, the sense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides in length.

[0223] In some embodiments, the sense strand and the antisense strand are each 15 to 30 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each 19 to 25 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each 21 to 23 nucleotides in length.

[0224] In some embodiments, one strand has at least one stretch of 1 to 5 single-stranded nucleotides in the double-stranded region. A "stretch of single-stranded nucleotides in a double-stranded region" means that there is at least one nucleotide base pair at both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. When both strands have a stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region, such single-stranded nucleotides can be opposite each other (e.g., a mismatched stretch) or can be positioned such that the second strand has no single-stranded nucleotides opposite the single-stranded iRNA of the first strand, and vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are located within 8 nucleotides of either end, e.g., 8, 7, 6, 5, 4, 3, or 2 nucleotides from the 5' or 3' end of the region of complementarity between the two strands.

[0225] In certain embodiments, the oligonucleotide comprises a single-stranded overhang on at least one end, hi certain embodiments, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0226] In some embodiments, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide-long single-stranded overhang at the 3' end.

[0227] In certain embodiments, each strand of the double-stranded iRNA has a ZXY structure as described in PCT Publication 2004080406, which is incorporated herein by reference in its entirety.

[0228] In some embodiments, the two strands of a double-stranded oligonucleotide can be linked to each other. The two strands can be linked to each other at both ends or only one end. When linked to one end, the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands are (N) n The oligonucleotides may be linked together by an oligonucleotide linker, including, but not limited to, (where N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, (where N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide). Some of the nucleotides in the linker may participate in base-pairing interactions with other nucleotides in the linker. The two strands may also be linked together by a non-nucleoside linker, such as a linker described herein. One of skill in the art will recognize that any of the oligonucleotide chemical modifications described herein, or variations thereof, can be used in the oligonucleotide linker.

[0229] Hairpin and dumbbell oligonucleotides have a duplex region of 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 or more nucleotide pairs. The duplex region can be up to 200, 100, or 50 nucleotide pairs in length. In certain embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0230] Hairpin oligonucleotides have a single-stranded overhang or terminal unpaired region, in some embodiments on the 3' and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhang is 1 to 4, more typically 2 to 3, nucleotides in length. Hairpin oligonucleotides capable of inducing RNA interference are also referred to herein as "shRNAs."

[0231] In certain embodiments, two oligonucleotide strands specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense strand to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments and under the conditions under which the assay is performed in the case of in vitro assays.

[0232] As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense strand hybridizes to its target sequence but hybridizes to a minimum number of other sequences. Stringent conditions are sequence-dependent and will vary under different circumstances; the "stringent conditions" under which an antisense strand hybridizes to a target sequence are determined by the nature and composition of the antisense strand and the assay being investigated.

[0233] It is understood in the art that the incorporation of nucleotide affinity modifications allows for a greater number of mismatches compared to unmodified oligonucleotides.Similarly, some oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, for example, by determining melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443).

[0234] Additive dsRNA design In some embodiments, the iRNA agent is a 19 nt long double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0235] In some embodiments, the iRNA agent is a 20 nt long double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0236] In some embodiments, the iRNA agent is a 21 nt long double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0237] In one embodiment, the iRNA agent comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the iRNA is blunt, while the other end comprises a 2-nt overhang. Preferably, the 2-nt overhang is at the 3' end of the antisense strand. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0238] In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length, and wherein positions 1 to 23 of the first strand, starting from the 5'-terminal nucleotide (position 1), comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions that pair with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and wherein the 5' end of the antisense strand is unpaired with the sense strand. the antisense strand comprises 10 to 30 consecutive nucleotides, thereby forming a 10 to 30 nucleotide single-stranded 5' overhang; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense strand and the antisense strand; and the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the antisense strand so as to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0239] In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, wherein the iRNA agent comprises a first strand having a length of at least 25 and a maximum of 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the second strand is 1 to 4 nucleotides longer at the 3' end than the first strand, wherein the at least 25-nucleotide long duplex region and the second strand are sufficiently complementary to a target mRNA along at least 19 nt of the second strand length to reduce target gene expression when the iRNA agent is introduced into a mammalian cell, wherein dicer cleavage of the iRNA preferentially results in an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0240] In some embodiments, the sense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site of the sense strand. For example, the sense strand can contain at least one motif of three 2'-F modifications on three consecutive nucleotides within positions 7-15 from the 5' end.

[0241] In some embodiments, the antisense strand may also contain at least one motif of three identical modifications of three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site of the antisense strand. For example, the antisense strand may contain at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides within positions 9-15 from the 5' end.

[0242] For iRNA agents having a duplex region 17-23 nt in length, the cleavage sites of the antisense strand are typically approximately positions 10, 11, and 12 from the 5' end. Thus, the three identically modified motifs can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide at the 5' end of the antisense strand or from the first paired nucleotide of the duplex region from the 5' end of the antisense strand. The cleavage site of the antisense strand can also vary depending on the length of the duplex region of the iRNA from the 5' end.

[0243] In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand comprises at least two motifs of three identical modifications of three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site of the strand, and at least one of the motifs occurs in another portion of the strand, away from the motif at the cleavage site by at least one nucleotide. In some embodiments, the antisense strand also comprises at least one motif of three identical modifications of three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site within the strand. The modification of the motif occurring at or near the cleavage site of the sense strand is different from the modification of the motif occurring at or near the cleavage site of the antisense strand.

[0244] In some embodiments, the iRNA agent includes a sense strand and an antisense strand each having 14 to 30 nucleotides, wherein the sense strand includes at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site of the strand. In some embodiments, the antisense strand also includes at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0245] In some embodiments, the iRNA agent includes a sense strand and an antisense strand each having 14 to 30 nucleotides, wherein the sense strand includes at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and wherein the antisense strand includes at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0246] In some embodiments, the iRNA agent contains mismatches with the target within the duplex or combinations thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their propensity to promote dissociation or melting (e.g., the free energy of association or dissociation of a particular pairing; the simplest approach is to test each pair individually, but next-neighbor or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C; G:U is preferred to G:C; and I:C is preferred to G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (as described elsewhere herein), are preferred to canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred to canonical pairings.

[0247] In certain embodiments, the iRNA agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs in the duplex region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0248] In some embodiments, the nucleotide at the first position in the double-stranded region from the 5'-end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5'-end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5'-end of the antisense strand is an AU base pair.

[0249] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the dsRNA agent is modified. For example, when a dsRNA agent is 50% modified, 50% of all nucleotides present in the dsRNA agent contain a modification described herein.

[0250] In certain embodiments, the oligonucleotide comprises one or more 2'-O modifications selected from the group consisting of 2'-deoxy, 2'-O-methoxyalkyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0251] In some embodiments, each of the sense and antisense strands is independently modified with a non-natural nucleotide, such as an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0252] In certain embodiments, the sense and antisense strands of the dsRNA agent each contain at least two different modifications.

[0253] In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. In one example, the oligonucleotide comprises 9 or 10 2'-F modifications.

[0254] In some embodiments, the oligonucleotide does not contain any 2'-F modifications.

[0255] The iRNA agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur at any nucleotide at any position in the sense strand, the antisense strand, or both strands. For example, the internucleotide linkage modification may occur at every nucleotide in the sense strand or the antisense strand; each internucleotide linkage modification may occur in an alternating pattern in the sense strand or the antisense strand; or the sense strand or the antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modifications in the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of the internucleotide linkage modifications in the sense strand may be shifted relative to the alternating pattern of the internucleotide linkage modifications in the antisense strand.

[0256] In some embodiments, the iRNA contains a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can contain two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between them. The internucleotide bond modification can also be used to link the overhang nucleotide to the terminal paired nucleotide in the duplex region. For example, at least two, three, four, or all of the overhang nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide bonds, and optionally, an additional phosphorothioate or methylphosphonate internucleotide bond can be present between the overhang nucleotide and the paired nucleotide next to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhang nucleotides and the third is the paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides can be located at the 3' end of the antisense strand.

[0257] In some embodiments, the sense strand and / or the antisense strand comprise one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0258] In some embodiments, the sense strand and the antisense strand each have 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0259] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand of the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0260] In some embodiments, the antisense strand of the dsRNA agent is 100% complementary to the target RNA to which it hybridizes and inhibits its expression through RNA interference. In other embodiments, the antisense strand of the dsRNA agent is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0261] In one embodiment, the present invention relates to a dsRNA agent, as defined herein, capable of inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermolabile nucleotide, wherein at least one of the thermolabile nucleotides occurs at or near a site opposite the seed region of the antisense strand (i.e., positions 2 to 8 at the 5' end of the antisense strand).

[0262] When the sense strand is 21 nucleotides long, the thermally destabilizing nucleotide is located, for example, at positions 14 to 17 of the 5' end of the sense strand. The antisense strand contains at least two modified nucleic acids smaller than the sterically bulky 2'-OMe modification. Preferably, the two modified nucleic acids smaller than the sterically bulky 2'-OMe modification are separated by 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0263] In certain embodiments, the dsRNA agent is (a) (i) 18–23 nucleotides long; (ii) three consecutive 2'-F modifications at positions 7 to 15; and a sense strand having (b) (i) 18–23 nucleotides long; (ii) at least a 2'-F modification anywhere in the strand; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); The antisense strand having wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more locations on at least one strand; and has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand; or has blunt ends at both ends of the duplex.

[0264] In certain embodiments, the dsRNA agent is (a) (i) 18–23 nucleotides long; (ii) less than four 2'-F modifications a sense strand having (b) (i) 18–23 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); antisense strand having wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more locations on at least one strand; and has a two-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand; or has blunt ends at both ends of the duplex.

[0265] In certain embodiments, the dsRNA agent is (a) (i) 19–35 nucleotides long; (ii) less than four 2'-F modifications a sense strand having (b) (i) 19–35 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5′ end); antisense strand wherein the duplex region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); and wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more locations on at least one strand; and has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or has blunt ends at both ends of the duplex.

[0266] In certain embodiments, a dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides of the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agent has less than 20%, less than 15%, and less than 10% non-naturally occurring nucleotides.

[0267] In certain embodiments, a dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides of the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% natural nucleotides, such that 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides.

[0268] In certain embodiments, a dsRNA agent comprises a sense strand and an antisense strand having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides of the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agent has 0% naturally occurring nucleotides, such that 2'-OH, 2'-deoxy, and 2'-OMe are naturally occurring nucleotides.

[0269] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand having 14-30 nucleotides, wherein the sense strand sequence is represented by Formula (I): [ka] [During the ceremony, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a represents an oligonucleotide sequence containing independently 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b represents an oligonucleotide sequence containing independently 1, 2, 3, 4, 5, or 6 modified nucleotides; each n p and n q independently represent overhanging nucleotides; where N b and Y do not have the same modification; where XXX, YYY and ZZZ each independently represent a motif of three identical modifications of three consecutive nucleotides. wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the antisense strand of the dsRNA comprises two blocks of 1, 2, or 3 phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0270] Various publications describe multimeric iRNAs and can be used in the iRNAs of the present invention. Such publications include WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, all of which are incorporated herein by reference in their entirety.

[0271] In some embodiments, the antisense strand is 100% complementary to the target RNA to which it hybridizes and inhibits its expression through RNA interference. In other embodiments, the antisense strand is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0272] Nucleic acid modification In some embodiments, the oligonucleotide comprises at least one nucleic acid modification described herein. For example, at least one modification is selected from the group consisting of a modified internucleoside linkage, a modified nucleic acid base, a modified sugar, and any combination thereof. Without limitation, such modifications can be present anywhere in the oligonucleotide. For example, the modifications can be present in one of the RNA molecules.

[0273] Nucleic acid modifications (nucleobases) The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. For nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3'→5' phosphodiester linkage.

[0274] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for use in the oligonucleotides described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nuvalalin, isoguanisine, or tubercidin) and any of the oligomeric modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is replaced with an unnatural and / or universal base, the nucleotide is said to contain a modified nucleobase and / or nucleobase modification as described herein. Modified nucleobases and / or nucleobase modifications can also include conjugate moieties, such as natural, unnatural, and universal bases, including ligands described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0275] The oligonucleotides described herein can also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Examples of modified nucleobases include other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nuvalalin, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6-(Isopentenyl)adenine, 6-(Alkyl)adenine, 6-(Methyl)adenine, 7-(Deaza)adenine, 8-(Alkenyl)adenine, 8-(Alkyl)adenine, 8-(Alkynyl)adenine, 8-(Amino)adenine, 8-(Halo)adenine, 8-(Hydroxyl)adenine, 8-(Thioalkyl)adenine, 8-(Thiol)adenine, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6 -(Dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine cytosine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil Uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1 ,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nuvalalin, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl phenyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N, 2 -substituted purines, N6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" may be used.

[0276] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an iRNA duplex. Some examples of universal nucleobases include 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl- These include, but are not limited to, imidazopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-propynylisocarbostyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0277] Further nucleobases include those disclosed in U.S. Patent 3,687,808; those disclosed in International Application PCT / US09 / 038425, filed March 26, 2009; those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; those disclosed in English et al., Angewandte Chemie, International Edition, 1991, 30, 613; Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993. All of the above are incorporated herein by reference.

[0278] In some embodiments, modified nucleobase is the nucleobase that has a structure that is significantly similar to that of parent nucleobase, such as 7-deazapurine, 5-methylcytosine or G-clamp.In some embodiments, nucleobase mimics include more complex structures, such as tricyclic phenoxazine nucleobase mimics.The method for preparing the above-mentioned modified nucleobase is well known to those skilled in the art.

[0279] Nucleic acid modification (sugar) The oligonucleotides provided herein can contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers, including nucleosides or nucleotides with modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, including, but not limited to, the addition of a substituent, bridging two non-geminal ring atoms to form a locked nucleic acid, or a bicyclic nucleic acid. In some embodiments, the oligonucleotide contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers that are LNAs.

[0280] In some embodiments of the locked nucleic acid, the 2' position of the furanosyl is independently -[C(R1)(R2)] n -, -[C(R1)(R2)] n -O-, -[C(R1)(R2)] n -N(R1)-, -[C(R1)(R2)] n -N(R1)-O-, -[C(R1R2)] n -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -C(=NR1)-, -C(=NR1)- O-, -C(=O)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -O-, -Si(R1)2-, -S(=O) x - and -N(R1)-; where: x is 0, 1 or 2; n is 1, 2, 3 or 4; Each R and R is independently H, a protecting group, hydroxyl, C-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20is aryl, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, a substituted heteroaryl, a C5-C7 alicyclic radical, a substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and Each J1 and J2 is independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl or a protecting group.

[0281] In some embodiments, each of the linkers of the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -C(R1R2)-N(R1)-O-, or -C(R1R2)-ON(R1)-. In other embodiments, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or C1-C 12 It is alkyl.

[0282] Certain LNAs have been prepared and disclosed in the patent and scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO94 / 14226; WO2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Patents 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Allocation Publications 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807).

[0283] Also provided herein are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bond to form a bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Patents 6,268,490 and 6,670,461). The bond can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; in the case of an ethylene group at this position, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy(4'-CH2-O-2')LNA and other bicyclic sugar analogs exhibit extremely high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against degradation by 3'-exonucleases, and good solubility. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0284] A similarly described isomer of methyleneoxy(4'-CH2-O-2')LNA is alpha-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have superior stability against 3'-exonucleases. Alpha-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras that exhibit potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0285] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO98 / 39352 and WO99 / 14226.

[0286] Analogs of methyleneoxy (4'-CH2-O-2') LNA, phosphorothioate-methyleneoxy (4'-CH2-O-2') LNA, and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO99 / 14226). Furthermore, the synthesis of a novel conformationally restricted, high-affinity oligonucleotide analog, 2'-amino-LNA, has been described in the literature (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Additionally, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of duplexes with complementary RNA and DNA strands has been previously reported.

[0287] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2'-bridged) ENA; substituted sugars, particularly 2'-substituted sugars having 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents; and bicyclic modified sugars, including 4'-thio modified sugars. Sugars can also be replaced by, among other, sugar mimetic groups. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars are U.S. Patents 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,5 91,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO2005 / 121371.

[0288] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) n CH2CH2OR, n = 1-50; "locked" nucleic acids (LNAs) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms of the furanose ring, thereby forming a bicyclic ring system; O-amine or O-(CH2) n Amines (n=1-10, amine=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine, or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0289] "Deoxy" modifications include hydrogen (i.e., the deoxyribose sugar, particularly associated with single-stranded overhangs); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; including alkenyl and alkynyl optionally substituted, for example, with an amino function.

[0290] Other suitable 2' modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0291] The modification at the 2' position may be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent at C2' of the ribose in the same configuration as the 2'-OH of arabinose.

[0292] A sugar can contain two different modifications, e.g., gem modifications, at the same carbon of the sugar. The sugar group can contain one or more carbons with the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, an oligonucleotide can contain one or more monomers containing, for example, arabinose as the sugar, that contain an alpha linkage at the 1' position of the sugar, e.g., an alpha-nucleoside. A monomer can also have the opposite configuration at the 4' position, e.g., C5' and H4', or the substituents replacing them are interchanged. When C5' and H4', or the substituents replacing them, are interchanged, the sugar is said to be modified at the 4' position.

[0293] The oligonucleotides disclosed herein can also contain abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having other chemical groups in place of the nucleobase at C1'. See, for example, U.S. Patent 5,998,203, the contents of which are incorporated herein by reference in their entirety. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms. The oligonucleotides can also contain one or more sugars that are L-isomers, e.g., L-nucleosides. Modifications to the sugar group can also include substitution of the 4'-O with sulfur, an optionally substituted nitrogen, or a CH2 group. In some embodiments, the bond between C1' and the nucleobase is in the α configuration.

[0294] Sugar modifications can also include "acyclic nucleotides," which refers to any nucleotide in which there is no C—C bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In certain embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0295] In certain embodiments, the sugar modification is selected from the group consisting of 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl, 2'-O-CH2-(4'-C) (LNA), 2'-O-CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F where 2'-O-Me is in the arabinose configuration.

[0296] It is understood that when a particular nucleotide is linked to an adjacent nucleotide through its 2' position, the sugar modifications described herein can be located at the 3' position of the sugar for that particular nucleotide, e.g., the nucleotide linked through the 2' position. The 3' modification can be in a xylose configuration. The term "xylose configuration" refers to the placement of the substituent at the C3' of the ribose in the same configuration as the 3'-OH in a xylose sugar.

[0297] The hydrogen attached to C4' and / or C1' may be replaced by a linear or branched optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein the backbone of the alkyl, alkenyl, and alkynyl may contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), a phosphorus-containing bond, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, or optionally substituted cycloalkyl, wherein R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR. 11 , C.O.R. 11 , CO2R 11 , [ka] NR21 R 31 ,CONR 21 R 31 , CON(H)NR 21 R 31 , ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 , OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 , ON=CR 41 R 51 , SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocycles; R 21 and R 31 each occurrence independently represents hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 each occurrence independently represents hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R.11 or CO2R 11 or NR 11 R 11 ' and R 11 and R 11 is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In certain embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is replaced.

[0298] In some embodiments, C4' and C5' together form an optionally substituted heterocycle, preferably containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino, where M is independently at each occurrence an alkali metal or transition metal having an overall charge of +1; and Y is O, S, or NR', where R' is hydrogen or an optionally substituted aliphatic. Preferably, this modification is at the 5' end of the iRNA.

[0299] In some embodiments, the oligonucleotide comprises at least two regions of at least two consecutive monomers of the above formula. In some embodiments, the oligonucleotide comprises a gapped motif. In some embodiments, the oligonucleotide comprises at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In some embodiments, the oligonucleotide comprises at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0300] In some embodiments, the oligonucleotide has the formula: [ka] [In the formula, Bx is a heterocyclic base moiety.] and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (S)-cEt monomer(s) of the formula:

[0301] In some embodiments, the monomer comprises a sugar mimetic. In some such embodiments, the mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while the nucleobase is maintained for hybridization to a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some situations, the mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acad Res. 2000, 28:2911-14, incorporated by reference). Methods for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those skilled in the art.

[0302] Nucleic acid modification (sugar bond) Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming oligonucleotides. Such linking groups are also referred to as intersugar linkages. Two major classes of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-).

[0303] As noted above, a cyclic disulfide moiety is described herein that can be introduced into one or more of the phosphorus-containing internucleotide linkage groups of an oligonucleotide as a temporary protecting group. The remaining phosphorus-containing internucleotide linkage groups can also be modified using the methods described below.

[0304] Modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to natural phosphodiester linkages. In some embodiments, linkages with chiral atoms can be prepared as racemic mixtures, separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

[0305] The phosphate group of the linking group can be modified by replacing one of its oxygen atoms with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic degradation. Examples of modified phosphate groups include phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linkage can be replaced with S, Se, BR3 (R is hydrogen, alkyl, or aryl), C (i.e., alkyl, aryl, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, or aryl), or (R is optionally substituted alkyl or aryl). The phosphorus atom of an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the above atoms or atom groups makes the phosphorus atom chiral; in other words, the phosphorus atom in a phosphate group modified in this manner is a stereogenic center. The stereogenic phosphorus atom may be in the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

[0306] Phosphorodithioates have both non-bridging oxygens replaced with sulfur. The phosphorus center of phosphorodithioates is achiral, preventing the formation of oligonucleotide diastereomers. Therefore, without wishing to be bound by theory, modifications to both non-bridging oxygens that eliminate chiral centers, such as phosphorodithioate formation, may be desirable in that diastereomeric mixtures cannot be produced. Thus, the non-bridging oxygens can independently be either O, S, Se, B, C, H, N, or OR (where R is alkyl or aryl).

[0307] Phosphate linkers can also be modified by substitution of the bridging oxygen (i.e., the oxygen connecting the phosphate and sugar of a monomer) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at one or both linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, substitution with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, substitution with nitrogen is preferred.

[0308] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced with a non-phosphorus group, or in which the phosphate group is replaced with a non-phosphorus group, are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0309] In some embodiments, the phosphate group can be replaced with a non-phosphorus-containing connector, such as a dephospho linker. A dephospho linker is also referred to herein as a non-phosphodiester linker. Without wishing to be bound by theory, it is believed that because the charged phosphodiester group is the reactive center for nucleolytic degradation, its replacement with a neutral structural mimic enhances nuclease stability. Also, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which the charged phosphate group is replaced with a neutral moiety.

[0310] Moieties that replace the phosphate group include amide (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonic acid, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methylenecarbonylamino, methyl ... Examples of linkages include, but are not limited to, ethylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-O-P(O)-O-S-S-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5' and nonionic linkages containing mixed N, O, S and CH2 moieties, such as, for example, Carbohydrate See "Modifications in Antisense Research," YS Sanghvi and PD Cook, Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Examples of preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0311] Those skilled in the art will appreciate that in some circumstances, substitution of a non-bridging oxygen can enhance cleavage of the intersugar bond by the adjacent 2'-OH, and therefore, in many circumstances, modification of the non-bridging oxygen will require modification of the 2'-OH, e.g., a modification that does not participate in cleavage of the adjacent intersugar bond, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0312] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates in an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the Sp isomer, phosphorothioates in an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0313] In some embodiments, the oligonucleotide further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, including up to all) modified or non-phosphodiester linkage. In some embodiments, the oligonucleotide further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, including up to all) phosphorothioate linkage.

[0314] Oligonucleotides in which the phosphate linker and sugar are replaced with nuclease-resistant nucleoside or nucleotide surrogates are also contemplated. Without wishing to be bound by theory, the absence of a repeatedly charged backbone is thought to reduce binding to proteins that recognize polyanions (e.g., nucleases). Also, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce modifications in which the bases are anchored by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA), and backbone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

[0315] The oligonucleotides described herein may contain one or more asymmetric centers, thus giving rise to enantiomers, diastereomers and other stereoisomeric configurations which may be defined in terms of absolute stereochemistry as (R) or (S), such as for sugar anomers, or (D) or (L), such as for amino acids, etc. Included in the oligonucleotides are all such possible isomers, as well as their racemic and optically pure forms.

[0316] Nucleic acid modification (end modification) In certain embodiments, the oligonucleotide further comprises a phosphate or phosphate mimetic at the 5'-end of the antisense strand. In certain embodiments, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0317] In certain embodiments, the 5' end of the antisense strand does not contain a 5'-vinylphosphonate (VP).

[0318] The termini of an iRNA agent can be modified. Such modifications can be at one or both ends. For example, the 3' and / or 5' ends of an iRNA can be conjugated to a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecule, such as a protecting group (e.g., sulfur-, silicon-, boron-, or ester-based). The functional molecule can be attached to the sugar via a phosphate group and / or a linker. The terminal atom of the linker can be connected to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Alternatively, the linker can be connected to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

[0319] When a linker / phosphate functional molecule-linker / phosphate array is interposed between the two strands of a double-stranded oligonucleotide, this array can substitute for a hairpin loop in a hairpin-shaped oligonucleotide.

[0320] Terminal modifications useful for modulating activity include modification of the 5'-end of an iRNA with a phosphate or phosphate analog. In some embodiments, the 5'-end of an iRNA is phosphorylated or contains a phosphoryl analog. Examples of 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. 5'-end modifications can also be useful for stimulating or inhibiting a subject's immune system. In some embodiments, the 5'-end of an oligonucleotide is modified [ka] wherein W, X, and Y are each independently O or (wherein R is hydrogen, alkyl, or aryl), S, Se, BR3 (wherein R is hydrogen, alkyl, or aryl), BH3 - , C (i.e., alkyl, aryl, etc.), H, NR (R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl, or aryl); A and Z, in each occurrence, are each independently absent, O, S, CH, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone may contain one or more of O, S, SS, and NR (R is hydrogen, alkyl, aryl) internally and / or terminally; and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A replaces the oxygen attached to the 5' carbon of the sugar. When N is 0, W and Y, together with the P to which they are attached, can form an optionally substituted 5-8 membered heterocycle, where W and Y are each independently O, S, NR', or alkylene. Preferably, the heterocycle is substituted with aryl or heteroaryl. In certain embodiments, one or both of the hydrogens at the C5' of the 5'-terminal nucleotide are replaced with a halogen, eg, F.

[0321] Examples of 5' modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); 5'-alpha-thiotriphosphate; 5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5' modifications include 5'-alkyl phosphonates (R(OH)(O)PO-5', where R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)PO-5', where R = alkyl ether, e.g., methoxymethyl (CHOMe), ethoxymethyl, etc.); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'). An example of another 5' modification is when Z is alkyl, optionally substituted at least once, e.g., ((HO)(X)PO[-(CH) a -OP(X)(OH)-O] b -5', ((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5'; Dialkyl-terminated phosphates and phosphate mimetics; HO[-(CH2) a -OP(X)(OH)-O] b -5', H2N[-(CH2) a -OP(X)(OH)-O] b-5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5', where a and b are each independently 1 to 10. Other embodiments include BH3, BH3 - and / or containing substitution with Se.

[0322] Terminal modifications may also be useful for monitoring distribution, in which case preferred groups to add include fluorophores, such as fluorescein, or Alexa dyes, such as Alexa 488. Terminal modifications may also be useful for enhancing uptake, and modifications useful for this purpose include targeting ligands. Terminal modifications may also be useful for crosslinking to other portions of the oligonucleotide; modifications useful for this purpose include mitomycin C, psoralens, and their derivatives.

[0323] Thermal destabilization modification Oligonucleotides, such as iRNA or dsRNA agents, can be optimized for RNA interference by increasing the propensity of the iRNA duplex to separate or melt (decreasing the free energy of duplex association) by introducing a thermodestabilizing modification into the sense strand at a site opposite the seed region of the antisense strand (i.e., positions 2-8 at the 5' end of the antisense strand). This modification can increase the propensity of the duplex to separate or melt at the seed region of the antisense strand.

[0324] Thermally destabilizing modifications can include abasic modifications; mismatches with opposing nucleotides in opposing strands; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, e.g., Unlocked Nucleic Acids (UNA) or Glycerol Nucleic Acids (GNA).

[0325] Examples of abasic modifications are: [ka] is.

[0326] Examples of glycosylation are: [ka] is.

[0327] The term "UNA" refers to a non-locked acyclic nucleic acid, in which any of the sugar bonds has been removed to form a non-locked "sugar" residue. In one example, UNA also includes a monomer in which the C1'-C4' bond (i.e., the covalent carbon-oxygen carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Mikhailov et al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are incorporated herein by reference in their entirety). Acyclic derivatives offer greater backbone flexibility without affecting Watson pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0328] The term 'GNA' refers to glycol nucleic acid, a polymer similar to DNA or RNA but differing in the composition of its 'backbone', which consists of repeating glycerol units linked by phosphodiester bonds. [ka]

[0329] The heat destabilizing modification can be a mismatch (i.e., non-complementary base pair) between the heat destabilizing nucleotide and the opposite nucleotide of the opposite strand in the dsRNA duplex.Examples of mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatch base pairings known in the art are also suitable for the present invention.Mismatches can occur between nucleotides that are naturally occurring nucleotides or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases from each nucleotide regardless of the modification of the ribose sugar of the nucleotide.In some embodiments, oligonucleotides such as siRNA or iRNA agents contain at least one nucleobase in mismatch pairing that is a 2'-deoxynucleobase; for example, the 2'-deoxynucleobase is in the sense strand.

[0330] Further examples of abasic nucleotides, acyclic nucleotide modifications (UNA and GNA) and mismatch modifications are further detailed in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0331] Thermodestabilizing modifications can also include universal bases that have reduced or eliminated ability to form hydrogen bonds with opposing bases and phosphate modifications.

[0332] Nucleobase modifications that impair or completely abolish the ability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilizing the central region of the dsRNA duplex as described in WO2010 / 0011895, which is incorporated herein by reference in its entirety. Examples of nucleobase modifications include: [ka] is.

[0333] Examples of phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to the native phosphodiester bond are: [ka] is.

[0334] In some embodiments, oligonucleotides can contain 2'-5' linkages (2'-H, 2'-OH, and 2'-OMe, and P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or to prevent sense strand activation by RISC at the 5' end of the sense strand.

[0335] In other embodiments, oligonucleotides can contain L-sugars (e.g., L-ribose with 2'-H, 2'-OH, and 2'-OMe, L-arabinose). For example, these L-sugar modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0336] In some embodiments, one or more targeting ligands are connected to the modified phosphate prodrug compound, optionally via one or more linkers / tethers, via any of R2, R3, R4, R5, R6, R7, R8 and R9 of the [cyclic disulfide moiety].

[0337] Introduction of a targeting ligand into an oligonucleotide via the [cyclic disulfide moiety] of the sense strand, antisense strand, or both the sense and antisense strands is illustrated in Scheme 16 of Example 10 below. These targeting ligands can be cleaved at the [cyclic disulfide moiety] after the siRNA oligonucleotide enters the cytosol.

[0338] In some embodiments, the targeting ligand is selected from the group consisting of an antibody, a ligand-binding portion of a receptor, a ligand for a receptor, an aptamer, a carbohydrate-based ligand, a fatty acid, a lipoprotein, folate, thyrotropin, melanotropin, surfactant protein A, mucin, a glycosylated polyamino acid, transferrin, a bisphosphonic acid, a polyglutamic acid, a polyaspartic acid, a lipophilic moiety that enhances plasma protein binding, cholesterol, a steroid, a bile acid, vitamin B12, biotin, a fluorophore, and a peptide.

[0339] In some embodiments, at least one ligand is a carbohydrate-based ligand that targets liver tissue. In some embodiments, the carbohydrate-based ligand is selected from the group consisting of galactose, multivalent galactose, N-acetyl-galactosamine (GalNAc), multivalent GalNAc, mannose, multivalent mannose, lactose, multivalent lactose, N-acetyl-glucosamine (GlcNAc), multivalent GlcNAc, glucose, multivalent glucose, fucose, and multivalent fucose.

[0340] In certain embodiments, at least one ligand is a lipophilic moiety. In certain embodiments, the lipophilicity of the lipophilic moiety is determined by the log K ow or the hydrophobicity of the compound, as measured by the unbound fraction in a plasma protein binding assay of the compound, is greater than 0.2.

[0341] In some embodiments, the lipophilic moiety is a saturated or unsaturated C4-C 30 The lipophilic moiety comprises a hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety may be a saturated or unsaturated C6-C 18 Contains a hydrocarbon chain.

[0342] Further details regarding additional lipophilic moieties and the lipophilicity and hydrophobicity of lipophilic moieties of oligonucleotides can be found in PCT application PCT / US20 / 59399, entitled "Extrahepatic Delivery," filed November 6, 2020, the contents of which are incorporated herein by reference in their entirety.

[0343] In some embodiments, at least one ligand targets a receptor that mediates delivery to CNS tissue. In some embodiments, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bEnd.3 cell-binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter protein, and LDL receptor ligand.

[0344] In some embodiments, at least one ligand targets a receptor that mediates delivery to an ocular tissue. In some embodiments, the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, an LDL receptor ligand, and a carbohydrate-based ligand.

[0345] The targeting ligand can be introduced directly (independently, ie, not via the cyclic disulfide moiety) into the oligonucleotide.

[0346] In certain embodiments, the oligonucleotide comprises at least one targeting ligand at the 5' end, 3' end and / or an internal position of the antisense strand.

[0347] In certain embodiments, the oligonucleotide comprises at least one targeting ligand at the 5' end, 3' end and / or an internal position on the sense strand.

[0348] In one embodiment, the oligonucleotide comprises at least one cyclic disulfide moiety at the 5'-end, 3'-end and / or internal position of the antisense strand and at least one targeting ligand at the 5'-end, 3'-end and / or internal position of the sense strand.

[0349] In certain embodiments, the oligonucleotide comprises at least one cyclic disulfide moiety at the 5'-end of the antisense strand and at least one targeting ligand at the 3'-end of the sense strand.

[0350] In certain embodiments, one or more targeting ligands are connected to the modified phosphate prodrug compound (via the cyclic disulfide moiety) via one or more linkers / tethers, as described below.

[0351] In certain embodiments, one or more targeting ligands are attached directly to the oligonucleotide (ie, not via a cyclic disulfide moiety) via one or more linkers / tethers, as described below.

[0352] Linker / Tether The linker / tether connects to the modified phosphate prodrug compound at the "tethering attachment point (TAP)." The linker / tether can be any C1-C 100 Carbon-containing moieties (e.g., C1-C 75 , C1-C 50 , C1-C 20 , C1-C 10 ;C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 ) and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms a terminal amino or amido (NHC(O)-) group portion of the linker / tether that can serve as the attachment point for the modified phosphate prodrug compound. Non-limiting examples of linkers / tethers (underlined) include TAP -(CH 2 ) n NH- ;TAP- C(O)(CH 2 )n NH- ;TAP- NR''''(CH 2 ) n NH- , TAP- C(O)-(CH 2 ) n -C(O)- ;TAP- C(O)-(CH 2 ) n -C(O)O-;T AP- C(O)-O- ;TAP- C(O)-(CH 2 ) n -NH-C(O)- ;TAP- C(O)-(CH 2 ) n - ;TAP- C(O)-NH- ;TAP- C(O)- ;TAP- (CH 2 ) n -C(O)- ;TAP- (CH 2 ) n -C(O)O- ;TAP- (CH 2 ) n - ; or TAP- (CH 2 ) n -NH-C(O)- wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R"" is C1-C6 alkyl. Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen can form part of a terminal oxyamino group, e.g., -ONH2, or a hydrazino group, -NHNH2. The linker / tether can be optionally substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands include, for example, TAP -(CH 2 ) n NH (ligand) ;TAP- C(O)(CH 2 ) n NH (ligand);TAP- NR''''(CH 2 ) n NH (ligand) ;TAP -(CH 2 ) n ONH (ligand) ;TAP- C(O)(CH 2 ) n ONH (ligand) ;TAP- NR''''(CH 2 ) n ONH (ligand) ;TAP- (CH 2 ) n NHNH 2 (ligand) 、TAP- C(O)(CH 2 ) n NHNH 2 (ligand) ;TAP- NR''''(CH 2 ) n NHNH 2 (ligand) ;TAP- C(O)-(CH 2 ) n -C(O)(ligand) ;TAP- C(O)-(CH 2 ) n -C(O)O(ligand);T AP- C(O)-O(ligand) ;TAP- C(O)-(CH 2 ) n -NH-C(O)(ligand) ;TAP- C(O)-(CH 2 ) n (ligand) ;TAP- C(O)-NH(ligand) ;TAP- C(O)(ligand) ;TAP- (CH 2 ) n -C(O)(ligand) ;TAP- (CH 2 ) n -C(O)O(ligand) ;TAP- (CH 2 ) n (ligand) ;またはTAP- (CH 2 ) n -NH-C(O)(ligand)In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can be acylated, for example, with C(O)CF.

[0353] In some embodiments, the linker / tether can terminate in a mercapto group (i.e., SH) or an olefin (e.g., CH=CH). For example, the tether can be TAP -(CH 2 ) n -SH , TAP- C(O)(CH 2 ) n SH , TAP -(CH 2 ) n -(CH=CH 2 ) or TAP- C(O)(CH 2 ) n (CH=CH 2 ) where n can be as described elsewhere. The tether can be optionally substituted, for example, with hydroxy, alkoxy, perhaloalkyl, and / or can optionally contain one or more additional heteroatoms, for example, N, O, or S. The double bond can be cis or trans or E or Z.

[0354] In other embodiments, the linker / tether may contain an electrophilic moiety, preferably at the terminal position of the linker / tether. Examples of electrophilic moieties include, for example, aldehydes, alkyl halides, mesylates, tosylates, nosylates, or brosylates, or activated carboxylic acid esters, such as NHS esters or pentafluorophenyl esters. A preferred linker / tether (underlined) is TAP -(CH 2 ) n CHO ;TAP- C(O)(CH 2 ) n CHO ; or TAP- NR''''(CH 2 ) n CHO( where n is 1 to 6 and R'''' is C1-C6 alkyl); or TAP -(CH 2 ) n C(O)ONHS ;TAP- C(O)(CH 2 ) n C(O)ONHS ; or TAP- NR''''(CH 2 ) n C(O)ONHS (wherein n is 1 to 6 and R'''' is C1-C6 alkyl); TAP- (CH 2 ) n C(O)OC 6 F 5;TAP- C(O)(CH 2 ) n C(O)OC 6 F 5; or TAP- NR''''(CH 2 ) n C(O)OC 6 F 5 (wherein n is 1 to 11 and R'''' is C1-C6 alkyl); or -(CH 2 ) n CH 2 LG ;TAP- C(O)(CH 2 ) n CH 2 LG ; or TAP- NR''''(CH 2 ) n CH 2 LG (where n can be as described elsewhere and R"" is C1-C6 alkyl) (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be performed by coupling a nucleophilic group, e.g., a thiol or amino group, of the ligand with an electrophilic group on the tether.

[0355] In other embodiments, it may be desirable for the monomer to contain a phthalimide group (K) at the terminal position of the linker / tether. [ka]

[0356] In other embodiments, the other protected amino group can be at the terminal position of the linker / tether, e.g., alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety can be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0357] Any of the linkers / tethers described herein may also contain one or more additional linking groups, such as -O-(CH) n -, -(CH2) n -SS-, -(CH2) n - or -(CH=CH)-.

[0358] Cleavable Linker / Tether In certain embodiments, at least one of the linkers / tethers can be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0359] In certain embodiments, at least one of the linkers / tethers can be a reductively cleavable linker (eg, a disulfide group).

[0360] In certain embodiments, at least one of the linkers / tethers can be an acid-cleavable linker (eg, a hydrazone group, an ester group, an acetal group, or a ketal group).

[0361] In certain embodiments, at least one of the linkers / tethers can be an esterase-cleavable linker (eg, an ester group).

[0362] In certain embodiments, at least one of the linkers / tethers can be a phosphatase-cleavable linker (eg, a phosphate group).

[0363] In certain embodiments, at least one of the linkers / tethers can be a peptidase-cleavable linker (eg, a peptide bond).

[0364] Cleavable linkers are sensitive to cleaving factors, such as pH, redox activity, or the presence of degradative molecules. Generally, cleaving factors are found more frequently or at higher levels or activity within cells than in serum or blood. Examples of such degradative factors include, for example, oxidative or reductive enzymes or reducing agents, such as mercaptans present in cells that can degrade redox-cleavable linkers by reduction, which may be selective for a particular substrate or have no substrate specificity; esterases; agents that can create an endosomal or acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0365] Cleavable linking groups, such as disulfide bonds, can be pH sensitive. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH of 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some tethers have linking groups that cleave at a preferred pH, thereby liberating the iRNA agent from the ligand (e.g., a targeting or cell-permeable ligand, such as cholesterol) into the cell or desired cellular compartment.

[0366] The chemical bond (e.g., linking group) that links the ligand to the iRNA agent can include a disulfide bond. When the iRNA agent / ligand complex is taken up into a cell by endocytosis, the acidic environment of the endosome causes the disulfide bond to be cleaved, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. 19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002). The ligand can be a targeting ligand or a second therapeutic agent that can complement the therapeutic effect of the iRNA agent.

[0367] The tether may contain a linking group that can be cleaved by a specific enzyme. The type of linking group incorporated into the tether may depend on the cell targeted by the iRNA agent. For example, an iRNA agent targeting mRNA in hepatocytes may be conjugated to a tether containing an ester group. Hepatocytes are rich in esterases, and therefore the tether is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Cleavage of the tether may liberate the iRNA agent from the ligand attached to the distal end of the tether, thereby enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include lung, kidney cortex, and testicular cells.

[0368] A tether containing a peptide bond can be conjugated to an iRNA agent that targets peptidase-rich cell types, such as hepatocytes and synoviocytes. For example, an iRNA agent that targets synoviocytes, such as for the treatment of inflammatory diseases (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond.

[0369] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to withstand cleavage when contacted with blood or other non-target tissues, e.g., tissues to which the iRNA agent would be exposed when administered to a subject. Thus, the relative susceptibility to cleavage can be determined between a first condition and a second condition, where the first is selected to be indicative of cleavage in target cells and the second is selected to be indicative of cleavage in other tissues or body fluids, e.g., blood or serum. Evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0370] Redox-cleavable linking groups One class of cleavable linkers is redox-cleavable linkers, which are cleaved by reduction or oxidation. An example of a reduction-cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reduction-cleavable linker" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be attempted. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved up to 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0371] Phosphate-Based Cleavable Tethers Phosphate-based linking groups are linked to factors that decompose or hydrolyze phosphate groups. Examples of factors that cleave phosphate groups in cells include enzymes such as cellular phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0372] Acid-cleavable linking group An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less) or by an agent, such as an enzyme, that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when a carbon is attached to the oxygen of the ester (alkoxy group), an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0373] Ester-based linking groups Ester-based linking groups are cleaved by enzymes such as cellular esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0374] Peptide-based cleaving groups Peptide-based linking groups are cleaved by enzymes such as cellular peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linking groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a specific type of amide bond formed between amino acids that give rise to peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that give rise to peptides and proteins, and do not include the entire amide functionality. Peptide cleavable linking groups have the general formula -NHCHR 1 C(O)NHCHR 2 C(O)—, where R 1 and R 2 are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0375] Biocleavable Linkers / Tethers Linker can also comprise the biocleavable linker, which is nucleotide and non-nucleotide linker or their combination, connects one or both strands of two independent siRNA molecules to produce two parts of molecule, for example, bis(siRNA).In some embodiments, the simple electrostatic or stacking interaction between two independent siRNA can be the linker.Non-nucleotide linker comprises the tether or linker derived from monosaccharide, disaccharide, oligosaccharide and their derivatives, aliphatic, alicyclic, heterocyclic and their combinations.

[0376] In certain embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0377] In some embodiments, the biocleavable carbohydrate linker can have 1 to 10 saccharide units with at least one anomeric bond that allows the connection of two siRNA units. When more than one saccharide is present, the units can be linked via 1-3, 1-4, or 1-6 sugar bonds or alkyl chains.

[0378] Examples of biocleavable linkers are: [ka] [ka] Includes.

[0379] Further description of biocleavable linkers can be found in PCT application PCT / US18 / 14213, entitled "Endosomal Cleavable Linkers," filed January 18, 2018, the contents of which are incorporated herein by reference in their entirety.

[0380] Carrier In some embodiments, one or more targeting ligands are connected to the modified phosphate prodrug compound (via a [cyclic disulfide moiety]) via one or more carriers described herein and optionally via one or more linkers / tethers described herein.

[0381] In some embodiments, one or more targeting ligands are attached directly (i.e., not via a cyclic disulfide moiety) to the oligonucleotide via one or more carriers described herein and, optionally, via one or more linkers / tethers described herein.

[0382] The carrier can be a cyclic group or an acyclic group.In some embodiments, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin.In some embodiments, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0383] A carrier can replace one or more nucleotides of an iRNA agent.

[0384] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the iRNA agent.

[0385] In other embodiments, carrier replaces the terminal nucleotide of sense strand or antisense strand.In some embodiments, carrier replaces the terminal nucleotide of 3'-end of sense strand, thereby functioning as the terminal cap protection of 3'-end of sense strand.In some embodiments, carrier is a cyclic group having amine, for example, carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl or decalinyl.

[0386] A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to as a ribose-replacement modified subunit (RRMS). The carrier may be a cyclic or acyclic moiety and comprises two "backbone attachment points" (e.g., hydroxyl groups) and a ligand. The targeting ligand may be directly attached to the carrier or indirectly attached to the carrier via an intervening linker / tether as described above. [ka]

[0387] The ligand-conjugated monomer subunit can be the 5' or 3' terminal subunit of an iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit can occupy an internal position, and both "W" groups can be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit can be present in an iRNA agent.

[0388] Sugar replacement-based monomers, e.g., ligand-conjugated monomers (cyclic) Cyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugate monomers, are also referred to herein as RRMS monomer compounds. The carriers may have the following general formula (LCM-2): 9 R 10 If so, R 1 or R 2 ;R 3 or R 4 ; or R 9 and R 10 (two points can be selected from two main chain attachment points, e.g., R 1 and R 4 or R 4 and R 9 The preferred tethering point is when X is CH2, and R 7 ;R 5 or R 6 Carriers are described below as being capable of being incorporated into the chain. Thus, the structure also includes points of attachment, e.g., R 1 or R 2 ;R 3 or R 4 ; or R 9 or R 10 (Y is CR 9 R 10It is understood that this also includes cases where one (if at a terminal position) or two (if at an internal position) of the R groups (where R is an integer from 0 to 10) are attached to a phosphate or modified phosphate, e.g., a sulfur-containing backbone. For example, one of the R groups listed above can be -CH-, where one bond is attached to the carrier and one is attached to a backbone atom, e.g., a linking oxygen or central phosphorus atom. [ka] [During the ceremony, X is N(CO)R 7 , N.R. 7 or CH2; Y is NR 8 ,O,S,CR 9 R 10 and; Z is CR 11 R 12 is or does not exist; R 1 , R 2 , R 3 , R 4 , R 9 and R 10 each of which is independently H, OR a or (CH2) n OR b However, R 1 , R 2 , R 3 , R 4 , R 9 and R 10 At least two of the following are OR a and / or (CH2) n OR b and; R 5 , R 6 , R 11 and R 12 each independently represents a ligand, H, and optionally 1 to 3 R 13 C1-C6 alkyl or C(O)NHR substituted with 7 or R 5 and R 11 is sometimes combined with R 14 is a C3-C8 cycloalkyl substituted with; R7 can be a ligand, e.g., R 7 is R d or R 7 is, for example, a tethering part, e.g., NR c R d C1-C substituted with 20 alkyl; or NHC(O)R d C1-C substituted with 20 It may be a ligand indirectly tethered to a support via an alkyl; R 8 is H or C1-C6 alkyl; R 13 is hydroxy, C1-C4 alkoxy or halo; R 14 is NR c R 7 and; R 15 is C1-C6 alkyl or C2-C6 alkenyl optionally substituted with cyano; R 16 is C1-C 10 is alkyl; R 17 is a liquid or solid phase supported reagent; L is -C(O)(CH2) q C(O)- or -C(O)(CH2) q S- and; R a is a protecting group, e.g., CAr3; (e.g., dimethoxytrityl group) or Si(X 5’ )(X 5” )(X 5”’ ), where (X 5’ ), (X 5” ) and (X 5”’ ) are as described elsewhere, R b is P(O)(O - )H, P(OR 15 )N(R 16 )2 or LR 17 and; R c is H or C1-C6 alkyl; R dis H or a ligand; Each Ar is independently a C-C alkyl group optionally substituted with C-C alkoxy. 10 is aryl; n is 1 to 4; and q is 0 to 4.

[0389] Examples of carriers include, for example, those in which X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is absent; or X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 or X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 or X is CH2; and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2) or indane ring system, e.g., X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C5 cycloalkyl (H, z=1).

[0390] In certain embodiments, the carrier may be based on a pyrroline ring system or a 4-hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 and Y is CR 9R 10 and Z does not exist (D). [ka] OFG 1 is preferably attached to a primary carbon attached to one of the carbons of the five-membered ring, e.g., an exocyclic alkylene group, e.g., a methylene group (-CH2OFG in D) 1 ). OFG 2 is preferably directly bonded to one of the carbon atoms of the five-membered ring (-OFG of D) 2 For pyrroline-based supports, -CH2OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; or -CH2OFG 1 may be attached to C-3, OFG 2 may be attached at C-4. In some embodiments, CH2OFG 1 and OFG 2 can be geminally substituted at one of the carbons. For 3-hydroxyproline-based carriers, -CH2OFG 1 may be attached to C-2, OFG 2 may be attached at C-4. Thus, pyrroline and 4-hydroxyproline-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted about a particular bond, e.g., due to the presence of a ring. Thus, CHOFG 1 and OFG 2 may be cis or trans to each other in any of the pairings detailed above. Thus, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2The centers carrying D may both have the R configuration; or both have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). The tethering attachment point is preferably nitrogen. Preferred examples of carrier D include: [ka]

[0391] In certain embodiments, the carrier may be based on a piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 is. [ka] OFG 1 is preferably attached to a primary carbon attached to one of the carbons of the six-membered ring, for example, an exocyclic alkylene group, for example, a methylene group (n=1) or an ethylene group (n=2) [E's -(CH2) n OFG 1 ]. OFG 2 is preferably directly attached to one of the carbon atoms of the six-membered ring (-OFG of E) 2 ). -(CH2) n OFG 1 and OFG 2 may be arranged in a geminal fashion on the ring, i.e., both groups may be attached to the same carbon, e.g., C-2, C-3, or C-4. Alternatively, -(CH) n OFG 1 and OFG 2 may be arranged in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-2; -(CH2)n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-3. Piperidine-based monomers may therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted about a particular bond, e.g., due to the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 may be cis or trans to each other in any of the pairings detailed above. Thus, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers bearing the may both have the R configuration; or both have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). The tethering attachment point is preferably nitrogen.

[0392] In certain embodiments, the carrier may be based on a piperazine ring system (F), e.g., X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or a morpholine ring system (G), e.g., X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 is. [ka] OFG 1is preferably attached to a primary carbon attached to one of the carbons of the six-membered ring, for example, an exocyclic alkylene group, for example, a methylene group (-CH2OFG of F or G) 1 ). OFG 2 is preferably directly attached to one of the carbon atoms of the six-membered ring (-OFG of F or G) 2 ) for both F and G, -CH2OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; or vice versa. 1 and OFG 2 can be geminally substituted on one of the carbons. Piperazine- and morpholine-based monomers can therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted about a particular bond, e.g., due to the presence of a ring. Thus, CHOFG 1 and OFG 2 may be cis or trans to each other in any of the pairings detailed above. Thus, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers bearing F and G may both have the R configuration; or both have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). R''' can be, for example, C1-C6 alkyl, preferably CH3. The tethering attachment points are preferably at the nitrogens of both F and G.

[0393] In certain embodiments, the carrier can be based on a decalin ring system, e.g., X is CH; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11together form a C6 cycloalkyl (H, z=2) or indane ring system, e.g., X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C5 cycloalkyl (H, z=1). [ka] OFG 1 is preferably attached to a primary carbon atom attached to one of C-2, C-3, C-4 or C-5, for example, an exocyclic methylene group (n=1) or an ethylene group (n=2) [H of -(CH2) n OFG 1 ]. OFG 2 is preferably directly attached to one of C-2, C-3, C-4 or C-5 (-OFG of H) 2 ). -(CH2) n OFG 1 and OFG 2 may be arranged in a geminal fashion on the ring, i.e., both groups may be attached at the same carbon, e.g., C-2, C-3, C-4, or C-5. Alternatively, -(CH) n OFG 1 and OFG 2 may be arranged in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-3; -(CH2) n OFG1 may be attached to C-4, OFG 2 may be attached to C-5; or -(CH2) n OFG 1 may be attached to C-5, OFG 2 may be attached to C-4. Decalin or indane-based monomers may therefore contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted about a particular bond, e.g., due to the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 may be cis or trans to each other in any of the pairings detailed above. Thus, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers bearing the may both have the R configuration; or both have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). In a preferred embodiment, the substituents at C-1 and C-6 are trans to each other. The tethering attachment point is preferably at C-6 or C-7.

[0394] Other carriers may include those based on 3-hydroxyproline (J). [ka] Therefore, -(CH2) n OFG 1 and OFG 2 may be cis or trans to each other. Thus, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., CHOFG1 and OFG 2 (The centers bearing the may both have the R configuration; or both have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa). The tethering attachment point is preferably nitrogen.

[0395] Further details of representative cyclic, sugar-substituted based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0396] Sugar replacement-based monomers (acyclic) Acyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugate monomers, are also referred to herein as ribose-replaced monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers have the formula LCM-3 or LCM-4: [ka] may have:

[0397] In certain embodiments, each of x, y, and z, independently of one another, can be 0, 1, 2, or 3. In formula LCM-3, if y and z are different, the tertiary carbon can have an R or S configuration. In a preferred embodiment, in formula LCM-3, x is 0, y and z are each 1 (e.g., based on serinol), and in formula LCM-3, y and z are each 1. Formula LCM-3 or LCM-4 below can be optionally substituted, for example, with hydroxy, alkoxy, or perhaloalkyl.

[0398] Further details of representative acyclic, sugar-substituted based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entireties.

[0399] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to the 5' end of the sense strand or the 5' end of the antisense strand, optionally via a carrier and / or a linker / tether.

[0400] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to the 3' end of the sense strand or the 3' end of the antisense strand, optionally via a carrier and / or a linker / tether.

[0401] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to both ends of the sense strand, optionally via a carrier and / or a linker / tether.

[0402] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to both ends of the antisense strand, optionally via a carrier and / or a linker / tether.

[0403] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to an internal position of the sense or antisense strand, optionally via a carrier and / or linker / tether.

[0404] In some embodiments, one or more targeting ligands are conjugated to ribose, nucleobase and / or internucleotide bond.In some embodiments, one or more targeting ligands are conjugated to ribose at the 2', 3', 4' and / or 5' position of ribose.In some embodiments, one or more targeting ligands are conjugated to natural (e.g., A, T, G, C or U) or modified nucleobase as defined herein.In some embodiments, one or more targeting ligands are conjugated to phosphate or modified phosphate group as defined herein.

[0405] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to the 5' or 3' end of the sense strand and one or more identical or different targeting ligands conjugated to the 5' or 3' end of the antisense strand.

[0406] In some embodiments, at least one targeting ligand is located at one or more ends of sense strand or antisense strand.In some embodiments, at least one targeting ligand is located at the 3'-end or 5'-end of sense strand.In some embodiments, at least one targeting ligand is located at the 3'-end or 5'-end of antisense strand.

[0407] In some embodiments, at least one targeting ligand is conjugated to one or more internal positions of at least one strand. Internal positions of a strand refer to nucleotides at any position on the strand other than the 3'-end and 5'-end to end positions of the strand (e.g., two positions: excluding position 1 counting from the 3'-end and position 1 counting from the 5'-end).

[0408] In some embodiments, at least one targeting ligand is located at one or more internal positions of at least one strand, including all positions except the two terminal positions from each end of the strand (e.g., four positions: excluding positions 1 and 2 counting from the 3' end and positions 1 and 2 counting from the 5' end). In some embodiments, at least one targeting ligand is located at one or more internal positions of at least one strand, including all positions except the three terminal positions from each end of the strand (e.g., six positions: excluding positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).

[0409] In one embodiment, at least one targeting ligand is located at one or more positions at at least one end of the duplex region, including all positions of the duplex region, but not including the overhang region or the carrier replacing the terminal nucleotide at the 3' end of the sense strand.

[0410] In certain embodiments, at least one targeting ligand is located on the sense strand within the first 5, 4, 3, 2, or first base pairs at the 5' end of the antisense strand of the duplex region.

[0411] In certain embodiments, at least one targeting ligand (e.g., lipophilic moiety) is located at one or more internal positions of at least one strand other than the cleavage site region of the sense strand, e.g., the targeting ligand (e.g., lipophilic moiety) is not located at positions 9-12 counting from the 5'-end of the sense strand, e.g., the targeting ligand (e.g., lipophilic moiety) is not located at positions 9-11 counting from the 5'-end of the sense strand. Alternatively, the internal positions exclude positions 11-13 counting from the 3'-end of the sense strand.

[0412] In some embodiments, at least one targeting ligand (e.g., lipophilic moiety) is located at one or more internal positions of at least one of the antisense strands, excluding the cleavage site region of the antisense strand, e.g., internal positions excluding positions 12-14 counting from the 5' end of the antisense strand.

[0413] In some embodiments, at least one targeting ligand (e.g., a lipophilic moiety) is located at one or more internal positions of at least one strand, excluding positions 11-13 of the sense strand, counting from the 3' end, and positions 12-14 of the antisense strand, counting from the 5' end.

[0414] In certain embodiments, one or more targeting ligands (e.g., lipophilic moieties) are located at one or more of the following internal positions, counting from the 5' end of each strand: positions 4-8 and 13-18 of the sense strand and positions 6-10 and 15-18 of the antisense strand.

[0415] In certain embodiments, one or more targeting ligands (e.g., lipophilic moieties) are located at one or more of the following internal positions, counting from the 5' end of each strand: positions 5, 6, 7, 15, and 17 of the sense strand and positions 15 and 17 of the antisense strand.

[0416] Target gene Target genes for siRNA include, but are not limited to, genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, genes that express kinases, adaptor protein genes, genes that encode G protein superfamily molecules, genes that encode transcription factors, genes that mediate angiogenesis, viral genes, genes required for viral replication, cellular genes that mediate viral function, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes that mediate unwanted immune responses, genes that mediate pain processing, genes that mediate neurological diseases, alleles of genes found in cells characterized by loss of heterozygosity, or one allele of a polymorphic gene.

[0417] Specific examples of target genes for siRNA include PCSK-9, ApoC3, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, PDGF beta gene; Erb-B gene, Src gene; CRK gene; GRB2 gene; RAS gene; MEKK gene; JNK gene; RAF gene; Erk1 / 2 gene; PCNA (p21) gene; MYB gene; c-MYC gene; JUN gene; FOS gene; BCL-2 gene; and cyclin. IL-1 D gene; VEGF gene; EGFR gene; cyclin A gene; cyclin gene; WNT-1 gene; beta-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; survivin gene; Her2 / Neu gene; topoisomerase I gene; topoisomerase II alpha gene; p73 gene; p21(WAF1 / CIP1) gene, p27(KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor gene; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, e.g., MLL-AF9, BCR / ABL fusion gene; TEL / AML1 fusion gene; EWS / FLI1 fusion gene; TLS / FUS1 fusion gene; PAX3 / FKHR fusion gene; AML1 / ETO fusion gene; alpha v-integrin gene; Flt-1 receptor gene; tubulin gene; human papillomavirus genes, genes required for human papillomavirus replication child, human immunodeficiency virus gene, gene required for human immunodeficiency virus replication, hepatitis A virus gene, gene required for hepatitis A virus replication, hepatitis B virus gene, gene required for hepatitis B virus replication, hepatitis C virus gene, gene required for hepatitis C virus replication, hepatitis D virus gene, gene required for hepatitis D virus replication, hepatitis E virus gene, gene required for hepatitis E virus replication, hepatitis F virus gene, gene required for hepatitis F virus replication, hepatitis G virus gene, gene required for hepatitis G virus replication, hepatitis H virus gene, gene required for hepatitis H virus replication, respiratory syncytial virus gene,Genes required for respiratory syncytial virus replication, Herpes simplex virus genes, Genes required for herpes simplex virus replication, Herpes cytomegalovirus genes, Genes required for herpes cytomegalovirus replication, Herpes Epstein-Barr virus genes, Genes required for herpes Epstein-Barr virus replication, Kaposi's sarcoma-associated herpesvirus genes, Genes required for Kaposi's sarcoma-associated herpesvirus replication, JC virus genes, Human genes required for JC virus replication, Myxovirus genes, Genes required for myxovirus gene replication, Rhinovirus genes, Genes required for rhinovirus replication, Coronavirus genes, Genes required for coronavirus replication, West Nile virus genes, Genes required for West Nile virus replication, St. Louis encephalitis genes, Genes required for St. Louis encephalitis virus replication, Tick-borne encephalitis virus genes, Genes required for tick-borne encephalitis virus replication, Murray Valley encephalitis virus genes, Genes required for Murray Valley encephalitis virus replication, Dengue virus genes, Genes required for dengue virus replication, Simian virus 40 genes, Genes required for simian virus 40 replication, Human T-cell lymphotropic virus genes, genes required for human T-cell lymphotropic virus replication, Moloney murine leukemia virus genes, genes required for Moloney murine leukemia virus replication, encephalomyocarditis virus genes, genes required for encephalomyocarditis virus replication, measles virus genes, genes required for measles virus replication, varicella-zoster virus genes, genes required for varicella-zoster virus replication, adenovirus genes, genes required for adenovirus replication, yellow fever virus genes, genes required for yellow fever virus replication, poliovirus genes, poliovirus replication genes required for replication, poxvirus genes, genes required for poxvirus replication, malaria parasite genes, genes required for replication of malaria parasite genes, Mycobacterium ulcerans genes, genes required for replication of Mycobacterium ulcerans, Mycobacterium tuberculosis genes, genes required for replication of Mycobacterium tuberculosis, Mycobacterium leprae genes, genes required for replication of Mycobacterium leprae, Staphylococcus aureus genes, genes required for replication of Staphylococcus aureus, Streptococcus pneumoniae genes, genes required for replication of Streptococcus pneumoniae, Streptococcus pyogenes genes,Genes required for Streptococcus pyogenes replication, Chlamydia pneumoniae genes, genes required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae genes, genes required for Mycoplasma pneumoniae replication, integrin genes, selectin genes, complement system genes, chemokine genes, chemokine receptor genes, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene, proplatelet basic protein gene, MIP-1I gene, MIP-1J gene, RANTES gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene These include, but are not limited to, genes, CMBKR2 gene, CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, genes for components of ion channels, genes for neurotransmitter receptors, genes for neurotransmitter ligands, amyloid family genes, presenilin genes, HD gene, DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene, SCA8 gene, alleles found in loss of heterozygosity (LOH) cells, monoallelic genes of polymorphic genes, and combinations thereof.

[0418] Loss of heterozygosity (LOH) can result in hemizygosity of sequences, e.g., genes, in both LOH groups. This can result in significant genetic differences between normal cells and disease-state cells, e.g., cancer cells, and can provide useful differentiation between normal cells and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in diploid cells but hemizygous in cells with LOH. Regions of LOH often contain other sequences, including genes whose loss promotes unwanted growth, e.g., tumor suppressor genes, and other genes, in some cases essential for normal function, e.g., growth. The methods of the present invention utilize, in part, the specific regulation of one allele of an essential gene with the compositions of the present invention.

[0419] In certain embodiments, the present invention provides oligonucleotides that modulate microRNAs.

[0420] CNS targeting In certain embodiments, the present invention provides oligonucleotides targeting APP for early-onset familial Alzheimer's disease, ATXN2 for spinocerebellar ataxia type 2 and ALS, and C9orf72 for amyotrophic lateral sclerosis and frontotemporal dementia.

[0421] In certain embodiments, the present invention provides oligonucleotides targeting TARDBP for ALS, MAPT (Tau) for frontotemporal dementia, and HTT for Huntington's disease.

[0422] In certain embodiments, the present invention provides oligonucleotides targeting SNCA for Parkinson's disease, FUS for ALS, ATXN3 for spinocerebellar ataxia type 3, ATXN1 for SCA1, genes for SCA7 and SCA8, ATN1 for DRPLA, MeCP2 for XLMR, prion diseases, recessive CNS disorders: PRNP for Lafora disease, DMPK for DM1 (CNS and skeletal muscle) and TTR for hATTR (CNS, eye and systemic).

[0423] Spinocerebellar degeneration (SCD) is a genetic brain dysfunction disorder. Dominantly inherited forms of SCD, such as SCA1-8, are devastating disorders with no disease-modifying treatments. Examples of targets include SCA2, SCA3, and SCA1.

[0424] Further details of these CNS targeting receptors and associated diseases can be found in PCT application PCT / US20 / 59399, entitled "Extrahepatic Delivery," filed November 6, 2020, the contents of which are incorporated herein by reference in their entirety.

[0425] In certain embodiments, the present invention provides oligonucleotides that target genes for diseases, including, but not limited to, age-related macular degeneration (AMD) (dry and wet), scattershot chorioretinopathy, dominant retinitis pigmentosa 4, Fuchs' dystrophy, hATTR amyloidosis, hereditary and sporadic glaucoma, and Stargardt's disease.

[0426] In some embodiments, the oligonucleotide targets VEGF for wet (or exudative) AMD.

[0427] In certain embodiments, the oligonucleotide targets C3 for dry (or non-exudative) AMD.

[0428] In some embodiments, the oligonucleotide targets the CFB for dry (or non-exudative) AMD.

[0429] In some embodiments, the oligonucleotide targets MYOC for glaucoma.

[0430] In certain embodiments, the oligonucleotide targets ROCK2 for glaucoma.

[0431] In certain embodiments, the oligonucleotide targets ADRB2 for glaucoma.

[0432] In certain embodiments, the oligonucleotide targets CA2 for glaucoma.

[0433] In some embodiments, the oligonucleotide targets CRYGC for cataracts.

[0434] In certain embodiments, the oligonucleotide targets PPP3CB for dry eye syndrome.

[0435] Ligand In some embodiments, oligonucleotides are further modified by the covalent attachment of one or more conjugate groups. Generally, conjugate groups modify one or more properties of the conjugated compound of the present invention, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in the chemical field and are attached directly or via an optional linking moiety or linking group to a parent compound such as an oligonucleotide. A list of preferred conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0436] In some embodiments, the oligonucleotides further comprise targeting ligands that target receptors that mediate delivery to specific CNS tissues. These targeting ligands may be conjugated in combination with lipophilic moieties to enable specific intrathecal and systemic delivery.

[0437] Examples of targeting ligands that target receptors that mediate delivery to CNS tissues are peptide ligands such as Angiopep-2, lipoprotein receptor-related protein (LRP) ligands, bEnd.3 cell-binding ligands; transferrin receptor (TfR) ligands (which can use the iron transport system in the brain and transport cargo to the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells, glial cells), glucose transporter proteins, and LDL receptor ligands.

[0438] In some embodiments, the oligonucleotide further comprises a targeting ligand that targets the receptor that mediates delivery to specific ocular tissues. These targeting ligands can be combined with lipophilic moieties and conjugated to enable specific ocular delivery (for example, intravitreal delivery) and systemic delivery. Examples of targeting ligands that target the receptor that mediates delivery to ocular tissues include lipophilic ligands, such as all-trans retinol (targeting retinoic acid receptors); RGD peptides (targeting retinal pigment epithelial cells), such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or cyclo(-Arg-Gly-Asp-D-Phe-Cys); LDL receptor ligands; and carbohydrate-based ligands (targeting posterior ocular endothelial cells).

[0439] Preferred conjugate groups applicable in the present invention are lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acid Res., 1992, 20, 533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acid Res., 1990, 18, 3777); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0440] Generally, a wide variety of ligands can be attached to the oligonucleotides described herein. Ligands can include naturally occurring molecules or recombinant or synthetic molecules. Examples of ligands 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, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MP EG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic group, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimeric polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine quaternary salt, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acid, transferrin, bisphosphonic acid, polyglutamic acid, polyaspartic acid, aptamer, asialofetuin, hyaluronan, procollagen, immunoglobulin (e.g., antibody), insulin, transferrin, albumin, sugar-albumin conjugate, intercalating agent (e.g., acridine), crosslinker (e.g., psoralen, mitomycin C), porphyrin (e.g., TPPC4, texaphyrin) , sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, 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)cholenoic acid, dimethoxytrityl or phenoxazine), peptides (e.g., alpha helical peptides, amphipathic peptides, RGD peptides, cell penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen, aspirin, vitamins, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu in tetraazamacrocycles, 3+ complexes), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin B, vitamin K, vitamin B, such as folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, p38 MAP kinase activators, NF-κB activators, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNF alpha), interleukin-1 beta, gamma interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high density lipoprotein (HDL), and cell permeabilizing agents (e.g., spiral cell permeabilizing agents).

[0441] Peptide and peptidomimetic ligands include naturally occurring or modified peptides, e.g., D- or L-peptides; α, β, or γ peptides; N-methylpeptides; azapeptides; peptides with one or more amide, i.e., peptide bonds replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands 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.

[0442] Examples of amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, eboyapeptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, Esculentinis-1, and caerin.

[0443] As used herein, the term "endosomolytic ligand" refers to a molecule that has endosomolytic properties. An endosomolytic ligand promotes lysis and / or transport of a composition of the invention, or a component thereof, from a cellular compartment, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular endoplasmic reticulum, into the cytoplasm of a cell. Some examples of endosomolytic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEI), linear and branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0444] Examples of endosomolytic / fusogenic peptides are AALEALAEALEALAEALEALAEAAAAGGC (GALA); AALAEALAEALAEALAEALAEALAAAAGGC (EALA); ALEALAEALEALAEA; GLFEAIEGFIENGWEGMIWDYG (INF-7); GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3); EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, where n is norleucine); LFEALLELLESLWELLLEA (JTS-1); GLFKALLKLLKSLWKLLLKA (ppTG1); GLFRALLRLLRSLWRLLLRA (ppTG20); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA); GLFFEAIAEFIEGGWEGLIEGC (HA); GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin); H5WYG; and CHK6HC.

[0445] Without wishing to be bound by theory, fusogenic lipids fuse with membranes, resulting in their destabilization. Fusogenic lipids typically contain a small head group and an unsaturated acyl chain. Examples of fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0446] Synthetic polymers with endosomolytic activity applicable to the present invention are described in U.S. Patent Application Publications 2009 / 0048410; 2009 / 0023890; 2008 / 0287630; 2008 / 0287628; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, the contents of which are incorporated herein by reference in their entireties.

[0447] Examples of cell-penetrating peptides include RQIKIWFQNRRMKWKK (penetratin); GRKKRRQRRRPPQC (Tat fragment 48-60); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence-based peptide); LLIILRRRIRKQAHAHSK (PVEC); GWTLNSAGYLLKINLKALAALAKKIL (transportan); KLALKLALKALKAALKLA (amphipathic model peptide); RRRRRRRRR (Arg9); KFFKFFKFFK (bacterial cell wall-penetrating peptide); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin); These include, but are not limited to, DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39); ILPWKWPWWPWRR-NH2 (indolicidin); AAVALLPAVLLALLAP (RFGF); AALLPVLLAAP (RFGF analog); and RKCRIVVIRVCR (bactenecin).

[0448] Examples of cationic groups are, for example, O-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, for example, O(CH2) n Amines (e.g., amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CH2CH2NH) nThese include, but are not limited to, protonated amino groups derived from CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0449] As used herein, the term "targeting ligand" refers to any molecule that enhances affinity for a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, for example, a cell, tissue, or organ compartment. Some examples of targeting ligands include, but are not limited to, antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0450] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3 (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent fucose, glycosylated polyamino acids, and lectins.The term multivalent indicates that there is more than one monosaccharide unit.These monosaccharide subunits are linked to each other via glycosidic bonds or to scaffold molecules.

[0451] Some folates and folate analogs applicable to the present invention as ligands are described in US Pat. Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the contents of which are incorporated herein by reference in their entireties.

[0452] As used herein, the terms "PK-modulating ligand" and "PK modulator" refer to a molecule that can modulate the pharmacokinetics of the compositions of the present invention. Some examples of PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, and transthyretin-binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligonucleotides containing several phosphorothioate intersugar linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, such as oligonucleotides containing approximately 5-30 nucleotides (e.g., 5-25 nucleotides, preferably 5-20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), and those containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., PK-modulating ligands) for the present invention. PK-modulating oligonucleotides can contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate and / or phosphorodithioate linkages. In certain embodiments, all internucleotide linkages in the PK-modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-modulating ligands for the present invention. Binding to serum components (eg, serum proteins) can be predicted from albumin binding assays such as those described in Oravcova, et al., Journal of Chromatography B (1996), 677: 1-27.

[0453] When two or more ligands are present, the ligands can all have the same properties, all have different properties, or some ligands have the same properties and others have different properties.For example, the ligands can have targeting properties, endosomolytic activity, or PK modulating properties.In a preferred embodiment, all ligands have different properties.

[0454] The ligand or tethered ligand may be present on the monomer when the monomer is incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention). In certain embodiments, the ligand may be incorporated by coupling to a "precursor" monomer after the "precursor" monomer has been incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention). For example, a monomer having an amino-terminated tether (i.e., no attached ligand), such as monomer-linker-NH, may be incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention). Subsequent to incorporation of the precursor monomer into a component of a compound of the invention (e.g., a compound or linker of the invention), a ligand having an electrophilic group, such as a pentafluorophenyl ester or aldehyde group, may then be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer tether.

[0455] In another example, a monomer bearing a chemical group suitable for oxidation in a click chemistry reaction can be incorporated, for example, into an azide- or alkyne-terminated tether / linker. Subsequent manipulations, i.e., after incorporation into the precursor monomer chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer via alkyne-azide coupling.

[0456] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety or internucleoside bond of oligonucleotide.The conjugation to purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms.In some embodiments, the 2nd, 6th, 7th or 8th position of purine nucleobase is bound to conjugated moiety.The conjugation to pyrimidine nucleobase or its derivative can also occur at any position.In some embodiments, the 2nd, 5th and 6th positions of pyrimidine nucleobase can be replaced with conjugated moiety.When the ligand is conjugated to nucleobase, the preferred position is not interfering with hybridization, i.e., not interfering with the hydrogen bond interaction required for base pairing.

[0457] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached to the conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to the conjugate moiety, such as at an abasic residue. Internucleoside linkages can also carry conjugate moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to the O, N, or S atom attached to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0458] There are several methods for preparing oligonucleotide conjugates.Generally, oligonucleotide is linked to conjugate moiety by the reactive group (for example, OH, SH, amine, carboxyl, aldehyde, etc.) of oligonucleotide and the reactive group of conjugate moiety.In some embodiments, one reactive group is electrophilic, and the other is nucleophilic.

[0459] For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or a thiol.Methods for conjugating nucleic acids and related oligonucleotides with and without linking groups are described in references such as Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0460] The ligand can be attached to the oligonucleotide via a linker or carrier monomer, e.g., a ligand carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "linkage attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally, a suitable bond available for incorporation into the backbone of the carrier monomer, e.g., a phosphate or modified phosphate of an oligonucleotide, e.g., a sulfur-containing backbone. A "linkage attachment point" (TAP) refers to an atom of the carrier monomer, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), that is connected to the selected moiety. The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the carrier monomer via an intervening tether. Thus, the carrier often contains a functional group, e.g., an amino group, or generally provides a bond suitable for incorporation or tethering of other chemical entities, e.g., a ligand, to the constituent atom.

[0461] Representative U.S. patents that teach conjugates of nucleic acids include U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,045; 77;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,149, 782;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241,5,391,723;5,416,203,5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587 ,371;5,595,726;5,597,696;5,599,923;5,599,928;5,672,662;5,688,941;5,714,166;6,153,737;6,172,208;6,300,319;6,335,434;6,335,437;6,395,437;6,444,806;6,486,308;6,525,031;6,528,631;6,559,279.

[0462] In some embodiments, the oligonucleotide further comprises a targeting ligand that targets liver tissue.In some embodiments, the targeting ligand is a carbohydrate-based ligand.In some embodiments, the targeting ligand is a GalNAc conjugate.

[0463] Because the ligand can be conjugated to the iRNA agent via a linker or carrier, and because the linker or carrier can include a branched linker, the iRNA agent can include multiple ligands via the same or different main chain attachment points to the carrier or via a branched linker. For example, the branch point of a branched linker can be a divalent, trivalent, tetravalent, pentavalent, or hexavalent atom or a group exhibiting such multiple valences. In some embodiments, the branch point is -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC; where Q is independently in each occurrence H or optionally substituted alkyl. In other embodiments, the branch point is glycerol or a glycerol derivative.

[0464] Evaluation of candidate iRNAs Candidate iRNA agents, such as modified RNAs, can be evaluated for a selected property by exposing the agent or modified molecule and a control molecule to appropriate conditions and evaluating the presence of the selected property. For example, resistance to degradation can be evaluated as follows: Candidate modified RNA (and a control molecule, usually in unmodified form) are exposed to degradative conditions, for example, to an environment containing a degradative agent, such as a nuclease. For example, a biological sample, such as one similar to the environment that may be encountered in therapeutic applications, can be used, for example, blood or a cell fraction, such as a cell-free homogenate or disrupted cells. The candidate and control can then be evaluated for resistance to degradation by any of several approaches. For example, the candidate and control can be labeled, for example, with a radioactive or enzymatic label or a fluorescent label, such as Cy3 or Cy5, before exposure. The control and modified RNA can be incubated with a degradative agent and, optionally, a control, for example, an inactivated, e.g., heat-inactivated, degradative agent. The physical parameters of the modified and control molecules, for example, size, can then be determined. The length of the molecule can be assessed by physical methods, such as polyacrylamide gel electrophoresis or sizing columns, to assess its native length or to assess functionality. Alternatively, Northern blot analysis can be used to assay the length of unlabeled modified molecules.

[0465] Functional assays can also be used to evaluate candidate drugs. Functional assays can be applied initially or after initial non-functional assays (e.g., assays for resistance to degradation) to determine whether modifications alter the ability of molecules to silence gene expression. For example, cells, such as mammalian cells, such as mouse or human cells, can be co-transfected with a plasmid expressing a fluorescent protein, such as GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, for example, WO00 / 44914). For example, modified dsiRNA homologous to GFP mRNA can be compared with control cells that are transfected but do not contain candidate dsiRNA, such as a no-drug control and / or an unmodified RNA control, to assess the ability to inhibit GFP expression by monitoring the decrease in cellular fluorescence. The effectiveness of candidate drugs on gene expression can be evaluated by comparing cellular fluorescence in the presence of modified and unmodified dssiRNA.

[0466] In another functional assay, candidate dssiRNA homologous to a maternally expressed gene, such as c-mos, can be injected into immature mouse oocytes to assess the ability of the drug to inhibit gene expression in vivo (see, e.g., WO01 / 36646). The oocyte phenotype, such as the ability to maintain metaphase II arrest, can be monitored as an indicator of the drug's inhibition of expression. For example, cleavage of c-mos mRNA by dssiRNA causes oocytes to terminate metaphase arrest and initiate parthenogenesis (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modifier on target RNA levels can be verified by Northern blot assays to assess a decrease in target mRNA levels or Western blot assays to assess a decrease in target protein levels, compared to a negative control. Controls can include cells to which no drug has been added and / or cells to which unmodified RNA has been added.

[0467] Physiological effects The siRNAs described herein can be designed to facilitate the determination of therapeutic toxicity through the complementarity of the siRNA with both human and non-human animal sequences. By these methods, the siRNA can be composed of a sequence that is completely complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human mammal, such as a rodent, ruminant, or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, bonobo, chimpanzee, rhesus monkey, or cynomolgus monkey. The siRNA sequence can be complementary to a sequence within a homologous gene in a non-human mammal and a human, such as an oncogene or tumor suppressor gene. Determining the toxicity of the siRNA in a non-human mammal can predict the toxicity of the siRNA in humans. For more stringent toxicity testing, the siRNA can be complementary to a human and more than one non-human animal, for example, two or three or more non-human animals.

[0468] The methods described herein can be used to correlate any physiological effect of siRNA in humans, for example, any unwanted effect, such as a toxic effect, or any positive or desired effect.

[0469] Increased cellular uptake of siRNA Described herein are various siRNA compositions that contain covalently linked conjugates that increase cellular uptake and / or intracellular targeting of siRNA.

[0470] Also provided are methods of the present invention, including administering siRNA and a drug that affects cellular uptake of siRNA. The drug may be administered before, after, or simultaneously with the siRNA. The drug may be covalently or non-covalently linked to the siRNA. The drug may be, for example, lipopolysaccharide, p38 MAP kinase activator, or NF-κB activator. The drug may have a transient effect on cells. The drug may increase cellular uptake of siRNA, for example, by disrupting the cellular cytoskeleton, for example, disrupting cellular microtubules, microfilaments, and / or intermediate filaments. The drug may be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The drug may also increase siRNA uptake into certain cells or tissues, for example, by activating an inflammatory response. Examples of drugs with such effects include tumor necrosis factor alpha (TNF alpha), interleukin-1 beta, CpG motifs, gamma interferon or more generally agents that activate Toll-like receptors.

[0471] siRNA production siRNAs can be produced, e.g., in large quantities, by a variety of methods, including organic synthesis and RNA cleavage, e.g., in vitro cleavage.

[0472] Organic Synthesis: siRNA can be produced by separately synthesizing each strand of a single-stranded RNA molecule or a double-stranded RNA molecule, and then annealing the component strands.

[0473] Large bioreactors, such as the OligoPilot II from Pharmacia Biotec AB (Uppsala, Sweden), can be used to produce large quantities of specific RNA strands of a given siRNA. The OligoPilot II reactor can efficiently couple nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. Ribonucleotide amidites are used to produce RNA strands. Standard cycles of monomer addition can be used to synthesize 21-23 nucleotide strands of siRNA. Typically, complementary strands are then produced separately and then annealed, for example, after release from the solid support and deprotection.

[0474] Organic synthesis can be used to produce individual siRNA species.The complementarity of this species to specific target gene can be precisely specified.For example, species can be complementary to the region that contains polymorphism, for example, single nucleotide polymorphism.Furthermore, the position of polymorphism can be precisely defined.In some embodiments, polymorphism is located in the internal region, for example, at least 4, 5, 7 or 9 nucleotides from one end or both ends.

[0475] dsiRNA cleavage. siRNA can also be produced by cleavage of larger siRNAs. Cleavage can be mediated in vitro or in vivo. For example, the following method can be used to produce iRNA by in vitro cleavage:

[0476] In vitro transcription. dsiRNAs are produced by transcription of nucleic acid (DNA) segments in both directions. For example, HiScribe TMThe RNAi transcription kit (New England Biolabs) provides a vector and method for producing dsiRNA from a nucleic acid segment cloned into the vector flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands of dsiRNA. The templates are transcribed in vitro by adding T7 RNA polymerase to produce dsiRNA. Similar methods using PCR and / or other RNA polymerases (e.g., T3 or SP6 polymerase) can be toxic and contaminate recombinant enzyme preparations.

[0477] In vitro cleavage.In some embodiments, the RNA produced by this method is, for example, by using Dicer or equivalent RNAse III-based activity to remove end siRNA in vitro and cleave into siRNA.For example, dsiRNA can be incubated in the in vitro extract from Drosophila or purified components, for example, purified RNAse or RISC complex (RNA-induced silencing complex).For example, see Ketting et al. Genes Dev 2001 Oct 15;15(20):2654-9; and Hammond Science 2001 Aug 10;293(5532):1146-50.

[0478] dsiRNA cleavage generally produces multiple siRNA species, each of which is a 21-23 nt fragment of the original dsiRNA molecule. For example, there may be siRNAs that contain complementary sequences in overlapping and adjacent regions of the original dsiRNA molecule.

[0479] Regardless of synthesis method, siRNA preparation can be prepared in a solution suitable for formulation (e.g., aqueous and / or organic solution).For example, siRNA preparation can be precipitated, redissolved in pure double-distilled water, and lyophilized.The dried siRNA can then be resuspended in a solution suitable for the intended formulation process.

[0480] Production of iRNA agents conjugated to targeting ligands In some embodiments, the targeting ligand is conjugated to the iRNA agent via a nucleobase, sugar moiety, or internucleoside linkage.

[0481] Conjugation to purine nucleobase or their derivatives can occur at any position, including endocyclic and exocyclic atoms.In some embodiments, the 2nd, 6th, 7th or 8th position of purine nucleobase is connected to conjugated moiety.Conjugation to pyrimidine nucleobase or their derivatives can also occur at any position.In some embodiments, the 2nd, 5th and 6th positions of pyrimidine nucleobase can be replaced with conjugated moiety.When targeting ligand is conjugated to nucleobase, preferred position is not interfering with hybridization, i.e., not interfering with the hydrogen bond interaction required for base pairing.In some embodiments, targeting ligand can be conjugated to nucleobase via the linker comprising alkyl, alkenyl or amide bond.

[0482] Conjugation to the sugar moiety of nucleoside can occur at any carbon atom.Examples of the carbon atom of the sugar moiety that can be bound to a targeting ligand include the 2', 3' and 5' carbon atoms.The targeting ligand can also be bound to the 1' position, such as at an abasic residue.In some embodiments, the targeting ligand can be conjugated to the sugar moiety via 2'-O modification, with or without a linker.

[0483] Internucleoside linkages can also carry targeting ligands. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the targeting ligand can be bound directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the targeting ligand can be bound to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0484] There are many methods for preparing oligonucleotide conjugate.Generally, oligonucleotide is linked to conjugate moiety by contacting the reactive group (for example, OH, SH, amine, carboxyl, aldehyde, etc.) of oligonucleotide with the reactive group of conjugate moiety.In some embodiments, one reactive group is electrophilic, and the other is nucleophilic.

[0485] For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or a thiol.Methods for conjugating nucleic acids and related oligonucleotides with and without linking groups are described in references such as Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0486] In some embodiments, a first (complementary) RNA strand and a second (sense) RNA strand can be synthesized separately, where one of the RNA strands comprises a pendant targeting ligand, and the first and second RNA strands can be mixed to form dsRNA. RNA strand synthesis preferably involves solid-phase synthesis, where individual nucleotides are joined end-to-end by forming internucleotide 3'-5' phosphodiester bonds in successive synthesis cycles.

[0487] In some embodiments, a targeting ligand having a phosphoramidite group is linked to the 3'-end or 5'-end of the first (complementary) or second (sense) RNA strand in the final synthesis cycle.In solid-phase synthesis of RNA, nucleotides are first in the form of nucleoside phosphoramidite.In each synthesis cycle, additional nucleoside phosphoramidites are linked to the -OH group of previously incorporated nucleotides.If a targeting ligand has a phosphoramidite group, it can be linked to the free OH end of the RNA previously synthesized in solid-phase synthesis in a similar manner to nucleoside phosphoramidites.Synthesis is carried out in an automated and standardized manner using a conventional RNA synthesizer.Synthesis of a targeting ligand having a phosphoramidite group can include phosphitylation of free hydroxyl to produce phosphoramidite groups.

[0488] Generally, oligonucleotides can be synthesized using protocols known from the literature, for example, Caruthers et al., Methods in Enzymology (1992) 211:3-19; WO 99 / 54459; Wincott et al., Nucl. Acids Res. (1995) 23:2677-2684; Wincott et al., Methods Mol. Bio., (1997) 74:59; Brennan et al., Biotechnol. Bioeng. (1998) 61:33-45, and U.S. Patent 6,001,311, each of which is incorporated herein by reference in its entirety. Generally, oligonucleotide synthesis involves conventional nucleic acid protection and coupling groups such as dimethoxytrityl at the 5'-end and phosphoramidite at the 3'-end. In a non-limiting example, small-scale synthesis is performed on an Expedite 8909 RNA synthesizer sold by Applied Biosystems, Inc. (Weiterstadt, Germany) using ribonucleoside phosphoramidites sold by ChemGenes Corporation (Ashland, Mass.). Alternatively, synthesis can be carried out by a 96-well plate synthesizer, such as that produced by Protogene (Palo Alto, Calif.), or by methods such as those described in Usman et al., J. Am. Chem. Soc. (1987) 109:7845; Scaringe, et al., Nucl. Acids Res. (1990) 18:5433; Wincott, et al., Nucl. Acids Res. (1990) 23:2677-2684; and Wincott, et al., Methods Mol. Bio. (1997) 74:59, each of which is incorporated herein by reference in its entirety.

[0489] The nucleic acid molecules of the invention may be synthesized separately and combined together after synthesis, for example, by ligation (Moore et al., Science (1992) 256:9923; WO 93 / 23569; Shabarova et al., Nucl. Acids Res. (1991) 19:4247; Bellon et al., Nucleosides & Nucleotides (1997) 16:951; Bellon et al., Bioconjugate Chem. (1997) 8:204); or by hybridization after synthesis and / or deprotection. The nucleic acid molecules may be purified by gel electrophoresis using conventional methods or by high performance liquid chromatography (HPLC; see Wincott et al., supra, incorporated herein by reference in its entirety) and resuspended in water.

[0490] Pharmaceutical Composition In certain embodiments, the present invention relates to a pharmaceutical composition comprising an iRNA (siRNA), e.g., a double-stranded iRNA or ssiRNA (e.g., a precursor, e.g., a DNA encoding a large siRNA or siRNA that can be processed into an ssiRNA, e.g., a double-stranded iRNA or ssiRNA or a precursor thereof), comprising a nucleotide sequence complementary, e.g., substantially and / or exactly complementary, to a target RNA. The target RNA may be a transcription product of an endogenous human gene. In certain embodiments, the siRNA is (a) 19-25 nucleotides in length, e.g., 21-23 nucleotides, (b) complementary to the endogenous target RNA, and optionally, (c) comprises at least one 3' overhang 1-5 nt in length. In certain embodiments, the pharmaceutical composition may be an emulsion, microemulsion, cream, jelly, or liposome.

[0491] In one example, the pharmaceutical composition comprises an iRNA (siRNA) mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In some embodiments, the liposomes are cationic liposomes.

[0492] In other embodiments, the pharmaceutical composition comprises an iRNA (siRNA), e.g., a double-stranded iRNA or ssiRNA (e.g., a precursor, e.g., a DNA encoding a large siRNA or siRNA that can be processed into ssiRNA, e.g., a double-stranded iRNA or ssiRNA or a precursor thereof), mixed with a topical penetration enhancer. In some embodiments, the topical penetration enhancer is a fatty acid. The fatty acid may be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-10 It may be an alkyl ester, a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof.

[0493] In other embodiments, the topical penetration enhancer is a bile salt, which can be cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether, or a pharmaceutically acceptable salt thereof.

[0494] In other embodiments, the penetration enhancer is a chelating agent, which can be EDTA, citric acid, salicylic acid, N-acyl derivatives of collagen, laureth-9, N-aminoacyl derivatives of beta-diketones, or mixtures thereof.

[0495] In other embodiments, the penetration enhancer is a surfactant, such as an ionic or non-ionic surfactant, which can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, perfluorochemical emulsion, or a mixture thereof.

[0496] In other embodiments, the penetration enhancer may be selected from the group consisting of unsaturated cyclic ureas, 1-alkylalkones, 1-alkenylazacycloalkanones, steroidal anti-inflammatory agents, and mixtures thereof. In yet other embodiments, the penetration enhancer may be a glycol, pyrrole, azone, or terpene.

[0497] In certain embodiments, the present invention relates to pharmaceutical compositions comprising iRNA (siRNA), e.g., double-stranded iRNA or ssiRNA (e.g., a precursor, e.g., DNA encoding a large siRNA or siRNA that can be processed into ssiRNA, e.g., a double-stranded iRNA or ssiRNA or a precursor thereof), in a form suitable for oral delivery. In certain embodiments, oral delivery can be used to deliver siRNA compositions to cells or regions of the gastrointestinal tract, such as the small intestine or colon (e.g., for treating colon cancer). The oral delivery form can be a tablet, capsule, or gel capsule. In certain embodiments, the siRNA of the pharmaceutical composition regulates the expression of a cell adhesion protein, regulates cell proliferation rate, or has biological activity against eukaryotic pathogens or retroviruses. In other embodiments, the pharmaceutical composition includes an enteric coating that substantially prevents dissolution of the tablet, capsule, or gel capsule in the mammalian stomach. In certain embodiments, the enteric coating is a coating. The coating can be acetate phthalic acid, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, or cellulose acetate phthalate.

[0498] In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

[0499] In another embodiment, the oral dosage form of the pharmaceutical composition includes an additive. In one example, the additive is polyethylene glycol. In another example, the additive is Precirol.

[0500] In other embodiments, the oral dosage form of the pharmaceutical composition comprises a plasticizer, which can be diethyl phthalate, triacetin, dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0501] In one embodiment, the present invention relates to a pharmaceutical composition comprising an iRNA (siRNA) and a delivery vehicle. In one embodiment, the siRNA is (a) 19-25 nucleotides in length, e.g., 21-23 nucleotides, (b) complementary to an endogenous target RNA, and, optionally, (c) Contains at least one 3' overhang 1 to 5 nucleotides in length.

[0502] In some embodiments, the delivery vehicle can deliver iRNA (siRNA), for example, double-stranded iRNA or ssiRNA (e.g., DNA encoding a precursor, e.g., a large siRNA or siRNA that can be processed into ssiRNA, e.g., double-stranded iRNA or ssiRNA, or a precursor thereof) to cells via a local administration route. The delivery vehicle can be a microscopic vesicle. In one example, the microscopic vesicle is a liposome. In some embodiments, the liposome is a cationic liposome. In another example, the microscopic vesicle is a micelle. In some embodiments, the present invention relates to a pharmaceutical composition comprising an injectable siRNA, for example, double-stranded iRNA or ssiRNA (e.g., DNA encoding a precursor, e.g., a large siRNA or siRNA that can be processed into ssiRNA, e.g., double-stranded iRNA or ssiRNA, or a precursor thereof). In some embodiments, the pharmaceutical composition in an injectable form comprises a sterile aqueous solution or dispersion and a sterile powder. In some embodiments, the sterile solution can comprise a diluent such as water; saline solution; fixed oil, polyethylene glycol, glycerin, or propylene glycol.

[0503] In some embodiments, the present invention relates to a pharmaceutical composition comprising an oral dosage form of iRNA (siRNA), e.g., a double-stranded iRNA or ssiRNA (e.g., a precursor, e.g., a DNA encoding a large siRNA or siRNA that can be processed into ssiRNA, e.g., a double-stranded iRNA or ssiRNA or a precursor thereof). In some embodiments, the oral dosage form is selected from the group consisting of a tablet, a capsule, and a gel capsule. In other embodiments, the pharmaceutical composition comprises an enteric material that substantially prevents dissolution of the tablet, capsule, or gel capsule in the mammalian stomach. In some embodiments, the enteric material is a coating. The coating can be acetate phthalic acid, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, or cellulose acetate phthalate. In some embodiments, the pharmaceutical composition in oral dosage form can comprise a penetration enhancer, e.g., a penetration enhancer described herein.

[0504] In another embodiment, the oral dosage form of the pharmaceutical composition includes an additive. In one example, the additive is polyethylene glycol. In another example, the additive is Precirol.

[0505] In other embodiments, the oral dosage form of the pharmaceutical composition comprises a plasticizer, which can be diethyl phthalate, triacetin, dibutyl sebacate, dibutyl phthalate, or triethyl citrate.

[0506] In one embodiment, the present invention relates to a pharmaceutical composition comprising a rectal dosage form of iRNA (siRNA), for example, a double-stranded iRNA or ssiRNA (e.g., a precursor, e.g., a DNA encoding a large siRNA or siRNA that can be processed into ssiRNA, e.g., a double-stranded iRNA or ssiRNA or a precursor thereof). In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

[0507] In some embodiments, the present invention relates to a pharmaceutical composition comprising a vaginal dosage form of iRNA (siRNA), such as a double-stranded iRNA or ssiRNA (e.g., a precursor, e.g., a DNA encoding a large siRNA or siRNA that can be processed into ssiRNA, e.g., a double-stranded iRNA or ssiRNA or a precursor thereof). In some embodiments, the vaginal dosage form is a suppository. In other embodiments, the vaginal dosage form is a foam, cream, or gel.

[0508] In some embodiments, the present invention relates to a pharmaceutical composition comprising iRNA (siRNA), e.g., double-stranded iRNA or ssiRNA (e.g., a precursor, e.g., DNA encoding a large siRNA or siRNA that can be processed into ssiRNA, e.g., double-stranded iRNA or ssiRNA or their precursors), in a pulmonary or nasal dosage form. In some embodiments, the siRNA is incorporated into particles, e.g., macroparticles, e.g., microspheres. The particles can be produced by spray drying, freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination thereof. The microspheres can be formulated as a suspension, powder, or implantable solid.

[0509] Treatment Methods and Delivery Routes Another aspect of the invention relates to a method of reducing expression of a target gene in a cell, comprising contacting the cell with an oligonucleotide, hi certain embodiments, the cell is an extrahepatic cell.

[0510] Another aspect of the invention relates to a method of reducing expression of a target gene in a subject, comprising administering to the subject an oligonucleotide.

[0511] Another aspect of the invention relates to a method of treating a subject having a CNS disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded iRNA agent of the invention, thereby treating the subject. Examples of CNS disorders that can be treated with the methods of the invention include Alzheimer's, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's, Parkinson's, spinocerebellar, prion, and Lafora.

[0512] Oligonucleotides can be delivered to subjects by various routes depending on the type of target gene and the type of disorder to be treated. In some embodiments, oligonucleotides are administered extrahepatically, such as by ocular administration (e.g., intravitreal administration) or intrathecal or intracerebroventricular administration.

[0513] In some embodiments, the oligonucleotide is administered intrathecally or intracerebroventricularly. By intrathecally or intracerebroventricularly administering the double-stranded iRNA agent, the method can reduce expression of the target gene in brain or spinal tissue, e.g., the cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[0514] In some embodiments, examples of target genes are APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK and TTR.To reduce the expression of these target genes in subjects, oligonucleotides can be administered directly to the eye (for example, intravitreally).By administering double-stranded iRNA agent intravitreally, the method can reduce the expression of target genes in ocular tissues.

[0515] For ease of explanation, the formulations, compositions, and methods in this section will be described largely with respect to modified iRNA. However, it is understood that these formulations, compositions, and methods can be practiced with other siRNAs, such as unmodified siRNAs, and such practice is within the scope of the present invention. Compositions containing iRNA can be administered to a subject by a variety of routes. Exemplary routes include intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

[0516] The iRNA molecules of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically contain one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0517] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

[0518] The route and site of administration can be selected to enhance targeting.For example, to target muscle cells, intramuscular injection into the target muscle would be a logical choice.Lung cells can be targeted by administering iRNA in aerosol form.Vascular endothelial cells can be targeted by coating a balloon catheter with iRNA and mechanically introducing DNA.

[0519] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

[0520] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs, or non-aqueous media, tablets, capsules, lozenges, or troches. For tablets, carriers that can be used include lactose, sodium citrate, and salts of phosphoric acid. Disintegrants such as various starches and lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid composition can be combined with emulsifying and suspending agents. If desired, certain sweeteners and / or flavoring agents can be added.

[0521] Compositions for intrathecal or intracerebroventricular or intrathecal administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[0522] Formulations for parenteral administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives. Intraventricular injection may be facilitated, for example, by the use of an intraventricular catheter connected to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic.

[0523] For ocular administration, ointments or droppable liquids can be delivered by ocular delivery systems known in the art, such as applicators or eyedroppers. Such compositions can contain hyaluronic acid, chondroitin sulfate, mucomimetic agents such as hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylcuronium chloride, and conventional amounts of diluents and / or carriers.

[0524] In some embodiments, administration of iRNA (siRNA), for example, double-stranded iRNA or ssiRNA composition, is parenteral, for example, intravenous (for example, as a bolus or as a diffuse infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracerebroventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.Administration can be by the subject or by another person, for example, a medical professional.Medicine can be provided in a metered dose or a dispenser that dispenses a metered dose.Selected delivery methods are further detailed below.

[0525] Intrathecal administration. In some embodiments, administration is by intrathecal injection (i.e., injection into the spinal fluid that bathes the brain and spinal cord tissue). Intrathecal injection of iRNA agents into the spinal fluid can be performed by bolus injection or via a minipump that can be implanted under the skin and provides regular and constant delivery of siRNA into the spinal fluid. Depending on the size, stability and solubility of the injected compound, molecules are delivered into the spinal cavity. The circulation of spinal fluid from the choroid plexus where they are produced, down around the spinal cord and dorsal root ganglia, then through the cerebellum, across the cortex, and to the arachnoid granulations where the fluid can exit to the CNS, allows the target to be reached throughout the CNS.

[0526] In some embodiments, intrathecal administration is via a pump. The pump can be a surgically implanted osmotic pump. In some embodiments, the osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration.

[0527] In some embodiments, the intrathecal administration is a pharmaceutical intrathecal delivery system comprising a reservoir containing a volume of drug and a pump arranged to deliver a portion of the drug contained in the reservoir. Further details of the intrathecal delivery system can be found in PCT / US2015 / 013253, filed January 28, 2015, which is incorporated herein by reference in its entirety.

[0528] The amount of iRNA agent to be injected intrathecally or intracerebroventricularly varies for each target gene, and the appropriate amount to be applied can be determined individually for each target gene. Typically, this amount ranges from 10 μg to 2 mg, preferably from 50 μg to 1500 μg, and more preferably from 100 μg to 1000 μg.

[0529] Rectal Administration The present invention also provides methods, compositions, and kits for rectal administration or delivery of the siRNAs described herein.

[0530] Thus, iRNA (siRNA), e.g., a double-stranded iRNA or ssiRNA described herein (e.g., a precursor, e.g., a DNA encoding a larger siRNA or siRNA that can be processed into an ssiRNA, e.g., a double-stranded iRNA or ssiRNA or a precursor thereof), e.g., a therapeutically effective amount of an siRNA described herein, e.g., an siRNA having a double-stranded region of less than 40, e.g., less than 30 nucleotides, and having one or two 1-3 nucleotide single-stranded 3' overhangs, can be administered rectally, e.g., from the rectum to the lower or upper colon. This approach is particularly useful for treating inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps or colon cancer.

[0531] The medication is delivered to the site in the colon by introduction of a dispensing device, for example, a flexible, camera-guided device similar to those used for colonoscopy or polyp removal, which contains the means for delivery of the medication.

[0532] Rectal administration of siRNA is by means of an enema. The siRNA enema can be dissolved in saline or a buffered solution. Rectal administration can also be by means of a suppository, which can contain other ingredients, such as additives, for example, cocoa butter or hydropropylmethylcellulose.

[0533] Ocular delivery. The iRNA agents described herein can be administered to ocular tissue. For example, the medicament can be applied to the ocular surface or nearby tissue, such as the inside of the eyelid. It can be applied topically, for example, as a spray, drops, eye wash, or ointment. Administration can be provided by the subject or another person, such as a medical professional. The medicament can be provided in a metered dose or a dispenser that dispenses a metered dose. The medicament can also be provided inside the eye and can be introduced by a needle or other delivery device that can be introduced to a selected area or structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

[0534] In some embodiments, the double-stranded iRNA agent can be administered directly to the eye by injection into ocular tissues, such as periocular, conjunctival, sub-Tenon, intracameral, intravitreal, intraocular, anterior or posterior juxtascleral, subretinal, subconjunctival, retrobulbar, or intracanalicular injection; directly to the eye using a catheter or other placement device, such as a retinal pellet, intraocular insert, suppository, or implant containing a porous, non-porous, or gelatinous substance; topical eye drops or ointment; or by a sustained release device implanted adjacent to the conjunctival sac or sclera (transscleral) or within the sclera (intrascleral) or intraocularly. Intracameral injection can be from the cornea into the anterior chamber so that the agent reaches the trabecular meshwork. Intracanalicular injection can be from Schlemm's canal or into the venous collecting duct that drains into Schlemm's canal.

[0535] In some embodiments, the double-stranded iRNA agent can be administered to the eye, e.g., into the vitreous cavity of the eye, by intravitreal injection, such as in a pre-filled syringe in a ready-to-inject form for use by a medical professional.

[0536] For ocular delivery, the double-stranded iRNA agent can be combined with ophthalmologically acceptable preservatives, cosolvents, surfactants, thickeners, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ocular suspension or solution. Solution formulations can be prepared by dissolving the conjugate in a physiologically acceptable isotonic aqueous buffer. In addition, the solution can contain an acceptable surfactant to help dissolve the double-stranded iRNA agent. Viscosity enhancers such as hydroxymethylcellulose, hydroxyethylcellulose, methylcellulose, and polyvinylpyrrolidone can be added to the pharmaceutical composition to improve the retention of the double-stranded iRNA agent.

[0537] To prepare a sterile ophthalmic ointment formulation, the double-stranded iRNA agent is combined with a preservative in a suitable vehicle, such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations can be prepared by suspending the double-stranded iRNA agent in a hydrophilic base prepared from a combination, such as CARBOPOL®-940 (BF Goodrich, Charlotte, NC), by methods known in the art.

[0538] Topical delivery. Any of the siRNAs described herein can be administered directly to the skin. For example, the drug can be applied topically directly or to a layer of the skin, for example, using a microneedle or series of microneedles that penetrate the skin but not, for example, the underlying muscle tissue. Administration of the siRNA composition can be local. Topical application is, for example, delivery of the composition to the dermis or epidermis of a subject. Topical administration can be in the form of a transdermal patch, ointment, lotion, cream, gel, droplets, suppository, spray, liquid, or powder. Compositions for topical administration can be formulated as liposomes, micelles, emulsions, or other lipophilic molecular assemblies. Transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.

[0539] For ease of explanation, the formulations, compositions, and methods in this section will be described largely in relation to modified iRNA. However, it will be understood that these formulations, compositions, and methods can be practiced with other siRNAs, e.g., unmodified siRNAs, and such practice is within the scope of the present invention. In certain embodiments, siRNAs, e.g., double-stranded iRNAs or ssiRNAs (e.g., precursors, e.g., DNA encoding larger siRNAs or siRNAs that can be processed into ssiRNAs, e.g., double-stranded iRNAs or ssiRNAs or their precursors), are delivered to a subject by topical administration. "Topical administration" refers to delivery to a subject by direct contact of the formulation with the surface of the subject. While the most common form of topical delivery is to the skin, the compositions disclosed herein can also be applied directly to other surfaces of the body, such as the eye, mucous membranes, surfaces of body cavities, or internal surfaces. As noted above, topical delivery is most commonly to the skin. This term encompasses several administration routes, including, but not limited to, topical and transdermal. These administration methods typically involve penetration of the skin's permeability barrier and efficient delivery to the target tissue or layer. Topical administration can also be used to mean penetration of the epidermis and dermis, ultimately achieving systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject or to specific dermal or dermal tissues.

[0540] As used herein, the term "skin" refers to the epidermis and / or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of skin is called the epidermis. The epidermis consists of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale, with the stratum corneum being the surface of the skin and the stratum basale being the deepest part of the epidermis. The epidermis is 50 μm to 0.2 mm thick, depending on the location on the body.

[0541] Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is composed primarily of collagen in the form of fibrous bundles. These collagen bundles provide support for, among other things, blood vessels, lymphatic capillaries, glands, nerve endings, and immunologically active cells.

[0542] One of the main functions of the skin as an organ is to control the entry of substances into the body. The main permeability barrier of the skin is provided by the stratum corneum, which is formed from multiple layers of cells in various culture states. The spaces between the cells of the stratum corneum are further filled with various lipids arranged in a lattice-like formation, which provide a seal to enhance the skin permeability barrier.

[0543] The permeability barrier provided by the skin is largely impermeable to molecules with a molecular weight greater than about 750 Da. To cross the equivalent barrier of the skin, large molecules must use mechanisms other than normal osmotic pressure.

[0544] There are several factors that determine the skin permeability of administered drugs.These factors include the characteristics of the skin to be treated, the characteristics of delivery agent, the interaction between drug and delivery agent and drug and skin, the dosage of the drug to be applied, the form of treatment and post-treatment regimen.In order to selectively target the epidermis and dermis, it may be possible to formulate the composition with one or more penetration enhancers that allow the drug to penetrate into preselected layers.

[0545] Transdermal delivery is a valuable administration route for lipid-soluble therapeutics. The dermis is more permeable than the epidermis, allowing for much more rapid absorption through abraded, burned, or exposed skin. Inflammation and other physiological conditions that increase blood flow to the skin also increase transdermal absorption. Absorption via this route can be enhanced by the use of an oily vehicle (rubbing) or one or more penetration enhancers. Another effective method for delivering the compositions disclosed herein via the transdermal route is skin hydration and the use of sustained-release topical patches. The transdermal route provides a potentially effective means for delivering the compositions disclosed herein for systemic and / or local treatment.

[0546] Additionally, iontophoresis (the use of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (the use of ultrasound to increase the absorption of various therapeutic agents through biological membranes, particularly the skin and cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics for administration location and retention at the administration site (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) can be useful methods for enhancing the transport of topically applied compositions through skin and mucosal sites.

[0547] The provided compositions and methods can also be used to test the function of various proteins and genes in cultured or preserved skin tissues in vitro and in animals. The present invention can therefore be applied to testing the function of any gene. The methods of the present invention can also be used therapeutically or prophylactically, for example, to treat animals known or suspected to have diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, erythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease, and viral, fungal, and bacterial infections of the skin.

[0548] Pulmonary delivery. Any of the siRNAs described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by introducing a delivery device into the pulmonary system, e.g., by introducing a delivery device capable of dispensing the medication. Some embodiments can use pulmonary delivery methods via inhalation. The medication is provided in a dispenser that delivers the medication in a form small enough to be inhaled, e.g., wet or dry. The device can deliver a metered amount of medication. The subject or another person can deliver the medication. Pulmonary delivery is effective not only for disorders that directly affect lung tissue, but also for disorders that affect other tissues. siRNA can be formulated as a liquid or non-liquid for pulmonary delivery, e.g., a powder, crystal, or aerosol.

[0549] For ease of explanation, the formulations, compositions, and methods in this section will be described mostly in relation to modified iRNA. However, it will be understood that these formulations, compositions, and methods can be performed with other siRNAs, e.g., unmodified siRNAs, and such practice is within the scope of the present invention. Compositions containing siRNAs, e.g., double-stranded iRNAs or ssiRNAs (e.g., precursors, e.g., DNA encoding larger siRNAs or siRNAs that can be processed into ssiRNAs, e.g., double-stranded iRNAs or ssiRNAs or their precursors), can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation of a dispersion by a patient, allowing the composition within the dispersion, e.g., iRNA, to reach the lungs, where it can be readily absorbed directly into the blood circulation via the alveolar region. Pulmonary delivery can be effective for both systemic and localized delivery to treat pulmonary diseases.

[0550] Pulmonary delivery can be achieved by various approaches, including the use of 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 can be used. One benefit of using an atomizer or inhaler is that the device is self-contained, minimizing the possibility of contamination. Dry powder dispersion devices, for example, deliver drugs that can be easily formulated as dry powders. iRNA compositions can be stably stored as lyophilized or spray-dried powders, either by themselves or in combination with a suitable powder carrier. Delivery of inhalable compositions can be mediated by a dose-timing element, which, when incorporated into the device, can include a timer, dose counter, time measurement device, or time indicator, allowing for patient dose tracking, compliance monitoring, and / or dosing guidance during aerosol medication administration.

[0551] The term "powder" refers to a composition consisting of finely dispersed solid particles that are free-flowing and can be easily dispensed into an inhalation device and then inhaled by a subject, allowing the particles to reach the lungs and penetrate the alveoli. Thus, a powder can be said to be "inhalable." For example, the average particle size is less than about 10 μm in diameter and has a relatively uniform spherical distribution. In some embodiments, the diameter is less than about 7.5 μm, and in some embodiments, less than about 5.0 μm. Typically, the particle size distribution is about 0.1 μm to about 5 μm in diameter, sometimes about 0.3 μm to about 5 μm.

[0552] The term "dry" means that the moisture content of the composition is about 10% by weight (% w) water or less, usually about 5% w or less, and in some cases less than about 3% w. A dry composition may be one in which the particles are readily dispersible in an inhalation device to form an aerosol.

[0553] The term "therapeutically effective amount" is the amount present in the composition necessary to provide a desired level of drug in the treated subject to obtain the expected physiological response.

[0554] The term "physiologically effective amount" is the amount delivered to a subject to obtain the desired prophylactic or therapeutic effect.

[0555] The term "pharmaceutically acceptable carrier" means that the carrier can be taken into the lungs without significant adverse toxicological effects thereon.

[0556] 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 adjusters 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.

[0557] 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. The carbohydrate group includes lactose, trehalose, raffinose, maltodextrin, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being used in some embodiments.

[0558] Additives that are minor components of the compositions of the present invention may be included to improve structural stability and powder dispersibility during spray drying. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, etc.

[0559] Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate may be used in some embodiments.

[0560] Pulmonary administration of micellar iRNA formulations can be achieved by a metered dose spray device with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.

[0561] Oral or nasal delivery. Any of the siRNAs described herein can be administered orally, for example, in tablets, capsules, gel capsules, lozenges, troches, or liquid syrups. Additionally, the compositions can be applied topically to oral surfaces.

[0562] Any of the siRNAs described herein can be administered intranasally. Nasal administration can be achieved by introducing a delivery device into the nose, for example, by introducing a delivery device that can decompose the medicament. Nasal delivery methods include spray, aerosol, liquid, for example, droplets, or topical administration to the nasal surface. The medicament is provided in a dispenser that delivers a medicament in a form small enough to be inhaled, for example, wet or dry. The medicament can be delivered in a metered dose. The subject or another person can deliver the medicament.

[0563] Nasal delivery is effective not only for disorders that directly affect nasal tissues, but also for disorders that affect other tissues. siRNA can be formulated in liquid or non-liquid form for nasal delivery, such as powder or crystal. As used herein, the term "crystal" refers to a solid that has the structure or characteristics of a crystal, i.e., a particle with a three-dimensional structure in which planes interact at a defined angle and have a regular internal structure. The composition of the present invention can have various crystalline forms. Crystal forms can be produced by various methods, including, for example, spray drying.

[0564] For ease of explanation, the formulations, compositions, and methods in this section will be described largely in relation to modified iRNA. However, it will be understood that these formulations, compositions, and methods can be implemented with other siRNAs, such as unmodified siRNAs, and such implementations are within the scope of the present invention. Both oral and nasal membranes offer advantages over other administration routes. For example, drugs administered through these membranes have a fast onset of action, provide therapeutic plasma levels, avoid the first-currency effects of hepatic metabolism, and avoid exposing the drug to the unfavorable gastrointestinal (GI) environment. An additional advantage is the ease of access to the membrane site, allowing the drug to be easily applied, localized, and removed.

[0565] In oral delivery, composition can target oral surface, for example, sublingual mucosa, including the membrane on the front surface of tongue and the buccal mucosa that constitutes the floor of the oral cavity or the inside of the cheek.Sublingual mucosa is relatively permeable, and therefore provides rapid absorption and acceptable bioavailability of many drugs.In addition, sublingual mucosa is convenient, acceptable and accessible.

[0566] The ability of a molecule to cross the oral mucosa is thought to correlate with molecular size, lipid solubility, and peptide / protein ionization. Small molecules under 1000 daltons are thought to cross the mucosa rapidly. As molecular size increases, permeability decreases rapidly. Lipid-soluble compounds are more permeable than non-lipid-soluble molecules. Absorption is greatest when the molecule is non-ionized or electronically neutral. Therefore, charged molecules are the greatest barrier to absorption through the oral mucosa.

[0567] The iRNA pharmaceutical composition can also be administered to the human oral cavity by spraying the mixed micellar pharmaceutical formulation and propellant into the oral cavity, rather than inhaling, from a metered-dose spray dispenser.In some embodiments, the dispenser is first shaken before spraying the pharmaceutical formulation and propellant into the oral cavity.For example, the pharmaceutical can be sprayed into the oral cavity or directly applied to the oral cavity surface, for example, in liquid, solid, or gel form.This administration is particularly desirable for treating inflammation of the oral cavity, for example, gums or tongue.For example, in some embodiments, buccal administration can be sprayed into the oral cavity, for example, without inhaling, from a dispenser that dispenses the pharmaceutical composition and propellant, for example, a metered-dose spray dispenser.

[0568] Embodiments of the invention also relate to methods of delivering oligonucleotides to the CNS by intrathecal or intracerebroventricular delivery or to ocular tissue by ocular delivery, eg, intravitreal delivery.

[0569] Some embodiments relate to methods of reducing expression of a target gene in a subject, comprising administering to the subject an oligonucleotide described herein. In some embodiments, the oligonucleotide is administered intrathecally or intracerebroventricularly (to reduce expression of the target gene in brain or spinal tissue). In some embodiments, the oligonucleotide is administered ocularly, e.g., intravitreally (to reduce expression of the target gene in ocular tissue).

[0570] The present invention is further illustrated by the following examples, which should not be construed as further limiting. All references, pending patent applications and published patents cited herein are hereby expressly incorporated by reference. [Example]

[0571] The present invention will now be generally described and more readily understood with reference to the following examples, which are included merely as illustrations of certain aspects and embodiments of the invention and are not intended to limit the invention.

[0572] Example 1. Synthesis of cyclic disulfide-modified phosphate prodrug derivatives Scheme 1 [ka] Compound 800: Trans-1,2-dithiane-4,5-diol (3.04 g, 20 mmol) was dissolved in anhydrous THF (30 mL) under an inert atmosphere and cooled in a water / ice bath. Sodium hydride, 60% dispersion in oil (0.84 g, 21 mmol) was added, and the mixture was stirred for 30 minutes. Iodomethane (3.7 mL, 60 mmol) was added, and the mixture was allowed to warm slowly to room temperature overnight. The reaction mixture was concentrated under reduced pressure to give a colorless liquid. The product was isolated by silica gel flash chromatography of the crude material (3.66 g) with an isocratic 30% ethyl acetate in...

Claims

1. Formula (I): [Cyclic disulfide moiety] - [phosphorus coupling group] (I) [wherein the [cyclic disulfide moiety] is: 【Chemistry 1】 where: R １ is O or S and is bonded to the P atom of the phosphorus coupling group; R ２ , R ４ , R ６ , R ７ , R ８ and R ９ each independently represents H, halo, OR １３ or alkylene-OR １３ , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R １４ )(R １５ )(R １６ ) or alkylene-C(R １４ )(R １５ )(R １６ ), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R ｓｕｂ may be substituted with a group; R ３ and R ５ each independently represents H, halo, OR １３ or alkylene-OR １３ , N(R')(R") or alkylene-N(R')(R"), alkyl, C(R １４ )(R １５ )(R １６ ) or alkylene-C(R １４ )(R １５ )(R １６ ), alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which optionally contains one or more R ｓｕｂ or R ３ and R ５ forms a second ring together with the adjacent carbon atom and two sulfur atoms; G is O, N(R'), S or C(R １４ )(R １５ ) and n is an integer from 0 to 6; R １３ is independently at each occurrence H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, each of which optionally contains one or more R ｓｕｂ may be substituted with a group; R １４ , R １５ and R １６ are each independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, N(R')(R"); R′ and R″ are each independently H, alkyl, alkenyl, alkynyl, aryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, each of which optionally contains one or more R ｓｕｂ may be substituted with a group; and R ｓｕｂ is independently at each occurrence halo, haloalkyl, alkyl, alkaryl, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, arylcarbonyl, acyloxy, cyano, or ureido. A compound having the structure:

2. In the [cyclic disulfide moiety], R １ is O; G is CH ２ and n is 0 or 1; R ２ , R ４ , R ６ , R ７ , R ８ and R ９ each independently represents H, halo, OR １３ Or C １ -C ６ Alkylene-OR １３ , N(R')(R") or C １ -C ６ Alkylene-N(R')(R"), C １ -C ６ alkyl, aryl, and heteroaryl, each of which optionally contains one or more R ｓｕｂ may be substituted with a group; R ３ and R ５ each independently represents H, halo, OR １３ Or C １ -C ６ Alkylene-OR １３ , N(R')(R") or C １ -C ６ Alkylene-N(R')(R"), C １ -C ６ alkyl, aryl, and heteroaryl, each of which optionally contains one or more R ｓｕｂ or R ３ and R ５ forms a second ring of 6 to 8 atoms together with the adjacent carbon atoms and two sulfur atoms; R １３ is independently H, C １ -C ６ alkyl, aryl, alkylcarbonyl or arylcarbonyl; and R′ and R″ are each independently H or C １ -C ６ The compound of claim 1, wherein the aryl group is an alkyl group.

3. [Cyclic disulfide moiety] 【Chemistry 2】 2. The compound of claim 1 having the structure:

4. [Cyclic disulfide moiety]: 【Chemistry 3】 2. The compound of claim 1 having the structure:

5. R ２ is optionally substituted aryl, the compound of claim 3 or 4.

6. R ２ is optionally substituted C １ - ６ 5. The compound of claim 3 or 4, which is alkyl.

7. [Cyclic disulfide moiety] is a compound of the following formula Ia), Ib) and II): Ia): 【Chemistry 4】 Ib): 【Chemistry 5】 II): 【Chemistry 6】 2. The compound of claim 1 having a structure selected from one of:

8. [Phosphorus coupling group]: 【Chemistry 7】 wherein: X １ and Z １ each independently represents H, OH, OM, or OR １３ , S.H., S.M., S.R. １３ , C(O)H, S(O)H, or alkyl, each of which optionally contains one or more R ｓｕｂ group, N(R')(R"), B(R １３ ) ３ , B.H. ３ － , Se; or D-Q, where D is independently at each occurrence absent, O, S, N(R'), alkylene, each of which optionally is one or more of R ｓｕｂ group, and Q is, independently at each occurrence, a nucleoside or an oligonucleotide; X ２ and Z ２ each independently represents N(R')(R"), OR １８ or D-Q, where D is independently at each occurrence absent, O, S, N, N(R'), alkylene, each of which optionally represents one or more of R ｓｕｂ group, and Q is independently at each occurrence a nucleoside or an oligonucleotide; Y １ is S, O or N(R'); M is an organic or inorganic cation; and R １８ is H or optionally one or more R ｓｕｂ is an alkyl substituted with a group; A compound according to any one of claims 1 to 3.

9. [Phosphorus coupling group] 【Chemistry 8】 wherein: X １ and Z １ are each independently OH, OM, SH, SM, C(O)H, S(O)H, optionally substituted with one or more hydroxy or halo groups; １ -C ６ alkyl or D-Q; D is independently at each occurrence absent, O, S, NH, or optionally substituted with one or more halo groups. １ -C ６ alkylene; and Y １ is S or O; A compound according to any one of claims 1 to 3.

10. X １ is OH or SH; and Z １ The compound of claim 9, wherein is DQ.

11. [Phosphorus coupling group] 【Chemistry 9】 wherein: X ２ is N(R')(R"); Z ２ Is X ２ , OR １８ or D-Q; R １８ is substituted with H or cyano １ -C ６ is alkyl; and R′ and R″ are each independently C １ -C ６ is alkyl, A compound according to any one of claims 1 to 3.

12. [Phosphorus coupling group] 【Chemistry 10】 12. The compound of claim 11 having a structure selected from the group consisting of:

13. 2. The compound of claim 1 having a structure selected from the group consisting of: 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】

14. The phosphorus coupling group has the structure (P-I), and the cyclic disulfide moiety -P(Y １ )(X １ )-but 【Chemistry 14】 9. The compound of claim 8 having a structure selected from the group consisting of:

15. One or more ligands are optionally linked, via one or more linkers, to the R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ and R ９ The compound of claim 1, wherein the compound is connected to any one of the following:

16. 16. The compound of claim 15, wherein the ligand is selected from the group consisting of an antibody, a ligand binding portion of a receptor, a ligand for a receptor, an aptamer, a carbohydrate-based ligand, a fatty acid, a lipoprotein, a folate, a thyrotropin, a melanotropin, a surfactant protein A, a mucin, a glycosylated polyamino acid, a transferrin, a bisphosphonic acid, a polyglutamic acid, a polyaspartic acid, a lipophilic moiety, cholesterol, a steroid, a bile acid, vitamin B12, biotin, a fluorophore, and a peptide.

17. [Cyclic disulfide moiety]-P(Y １ )(X １ )- is the structure 【Chemistry 15】 10. The compound of claim 9, having the formula:

18. [Cyclic disulfide moiety]-P(Y １ )(X １ )- is the structure 【Chemistry 16】 10. The compound of claim 9, having the formula:

19. [Cyclic disulfide moiety]-P(Y １ )(X １ )- is the structure 【Chemistry 17】 10. The compound of claim 9, having the formula:

20. [Cyclic disulfide moiety]-P(Y １ )(X １ )- is the structure 【Chemistry 18】 10. The compound of claim 9, having the formula:

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