Cyclic Disulfide-Modified Phosphate-Based Oligonucleotide Prodrug

Abstract

The present invention relates to a compound comprising a structure of formula (I): [cyclic disulfide moiety]-[linker coupling group] (I). Wherein the [cyclic disulfide moiety] is having the structure of JPEG2025522880000367.jpg70145. The present invention also relates to an oligonucleotide comprising one or more compounds comprising the structure of formula (I), wherein at least one [linker coupling group] comprises a nucleoside or an oligonucleotide. The present invention also relates to a pharmaceutical composition comprising the oligonucleotides described herein and to a method of reducing or inhibiting the expression of a target gene comprising 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 priority based on U.S. Provisional Application No. 63 / 357,050, filed on June 30, 2022, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application is filed with an array in XML format, which is hereby incorporated by reference in its entirety. The XML copy was created on June 29, 2023, named 29520.1510 - PCT__SL.xml, and has a size of 4,495,791 bytes.

[0003] Field of the Invention The present invention generally relates to the field of modified phosphate - based oligonucleotide prodrugs.

Background Art

[0004] Background Phosphate esters are important intermediates for the formation of nucleotides and their assembly into RNA and DNA. Inside cells, phosphate groups generally serve as regulatable leaving groups. Phosphate esters are charged at physiological pH and help bind phosphate esters to the active sites of enzymes. However, for a phosphate ester to bind to an enzyme, it may be difficult for the charged molecule to pass through the cell membrane other than by endocytosis, so it must first penetrate the membrane to approach the enzyme. This limitation can be alleviated with compounds having large, more lipophilic substituents.

[0005] Alternatively, the prodrug approach is studying to temporarily mask any negative charges of phosphate esters on oligonucleotides 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 under various stimuli in vivo to yield the active parent drug. The prodrug approach of masking the negative charges of the phosphate groups of oligonucleotides with cell-cleavable protecting / masking groups can provide several advantages, including, for example, enhanced cell penetration and avoidance or minimization of degradation in serum through cell sequestration, compared to the unprotected counterparts.

[0006] However, the prodrug approach remains a significant challenge, partly because the selection of the best masking group is difficult. For example, cell cleavage of the protecting group can often produce products that are considered inconvenient or have undesirable properties. Furthermore, the protecting group must balance enabling absorption in the intestine and enabling cleavage in the blood or target cells. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] Therefore, there is a continuing need to develop novel and improved modified phosphate prodrugs for masking internucleotide phosphate linkages of oligonucleotides for the production of effective and efficient oligonucleotide-based drugs for efficient in vivo delivery and improved in vivo efficacy of oligonucleotides. MEANS FOR SOLVING THE PROBLEMS

[0008] Abstract One aspect of the invention relates to a compound having the structure of formula (I) or a salt or stereoisomer thereof: [Cyclic disulfide moiety]-[Linker coupling group] (I). The [Cyclic disulfide moiety] has CHEMICAL STRUCTURE the structure of. Alternatively, the [Cyclic disulfide moiety] is [Chemistry] may have the structure. In these formulas, R1 is O or S and is bonded to the P atom of [linker coupling group]; [Chemistry] represents a bond to [linker coupling group]; R2, R4, R6, R7, R8 and R9 are each independently H, halo, CN or alkylene-CN, C(O)OR 13 or alkylene-C(O)OR 13 , S(O)OR 13 or alkylene-S(O)OR 13 , C(O)N(R’)(R”) or alkylene-C(O)N(R’)(R”), 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 may optionally be substituted with one or more R sub groups; R3 and R5 are each independently H, halo, CN or alkylene-CN, C(O)OR 13 or alkylene-C(O)OR 13 , S(O)OR 13 or alkylene-S(O)OR 13 , C(O)N(R’)(R”) or alkylene-C(O)N(R’)(R”), 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 ) is alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which may optionally be substituted with one or more R sub groups; or R3 and R5 together with the adjacent carbon atoms and two sulfur atoms form a second ring; R2 and R3 together with the adjacent carbon atoms may form an additional ring; R4 and R5 together with the adjacent carbon atoms may form an additional ring; R6 and R7 together with the adjacent carbon atoms may form an additional ring; R8 and R9 together with the adjacent carbon atoms may form an additional ring; Two or more of R2, R3, R4, R5, R6, R7, R8, R9, R 14 and R 15 may together with adjacent carbon atoms form one or more rings fused to a ring containing two sulfur atoms; G is O, N(R’), S or C(R 14 )(R 15 ); n is an integer from 0 to 6; R 13 is in each case independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl or arylcarbonyl, each of which may optionally be substituted with one or more R sub groups; R 14 and 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, heteroaryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl or ω-hydroxyalkynyl, each of which may optionally be substituted with one or more R sub groups; or R’ and R” together with the adjacent nitrogen atom form a ring; and R sub is in each case independently halo, haloalkyl, alkyl, alkaryl, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, arylcarbonyl, acyloxy, cyano or ureido.

[0009] In certain embodiments, 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, CN or C1-C6 alkylene-CN, C(O)OR 13 or C1-C6 alkylene-C(O)OR 13 , S(O)OR 13 or C1-C6 alkylene-S(O)OR 13 , C(O)N(R’)(R”) or C1-C6 alkylene-C(O)N(R’)(R”), 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 may optionally be substituted with one or more R sub groups; R3 and R5 are each independently H, halo, CN or C1-C6 alkylene-CN, C(O)OR 13 or C1-C6 alkylene-C(O)OR 13 , S(O)OR 13 or C1-C6 alkylene-S(O)OR 13 , C(O)N(R’)(R”) or C1-C6 alkylene-C(O)N(R’)(R”), 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 may optionally be substituted with one or more R sub groups; or R3 and R5 together with adjacent carbon atoms and two sulfur atoms form a second ring of 6 to 8 atoms; R2 and R3 may together with adjacent carbon atoms form an additional ring of 3 to 7 atoms; R4 and R5 may together with adjacent carbon atoms form an additional ring of 3 to 7 atoms; R6 and R7 may together with adjacent carbon atoms form an additional ring of 3 to 7 atoms; R8 and R9 may together with adjacent carbon atoms form an additional ring of 3 to 7 atoms; Two or more of R2, R3, R4, R5, R6, R7, R8, R9, R 14 and R 15 may together with adjacent carbon atoms form one or more rings of 5 to 7 atoms fused to a ring containing two sulfur atoms; R 13 is in each case independently H, C1-C6 alkyl, aryl, alkylcarbonyl or arylcarbonyl; and R’ and R” are each independently H or C1-C6 alkyl.

[0010] In one embodiment, [the cyclic disulfide moiety] is

Chemical formula

[0011] In certain embodiments, [the cyclic disulfide moiety] is [Chemical formula] It has the structure. R2 can be an aryl optionally substituted, for example, phenyl optionally substituted. In certain embodiments, R2 is mono-, di- or tri-substituted phenyl. In certain embodiments, R2 is para-substituted phenyl. In certain embodiments, R2 is C1-6 alkyl optionally substituted. In certain embodiments, R2 is halo C1-6 alkyl. In certain embodiments, R2 is C 1-6 alkyl.

[0012] In certain embodiments, [the cyclic disulfide moiety] has a structure selected from the following groups Ia), Ib) and II). Group Ia) includes the following structures: [Chemical formula] Group Ib) includes the following structures: [Chemical formula] Group II) includes the following structures: [Chemical formula]

[0013] In certain embodiments, [the cyclic disulfide moiety] is [Chemical formula] It has the structure. R4 and R5 can each independently be H, C1-6 alkyl or phenyl. In certain embodiments, R4 and R5 are each independently C1-3 alkyl. In certain embodiments, R4 and R5 are each methyl. In certain embodiments, one or both of R4 and R5 are phenyl. R2 and R3 are as defined above. In certain embodiments, R2 and R3 are each independently H, C1-6 alkyl, CN or CH2CN, C(O)OR 13 or CH2C(O)OR 13 , S(O)OR 13 or CH2S(O)OR 13 , C(O)N(R’)(R”) or CH2C(O)N(R’)(R”) or C(R 14 )(R 15 )(R 16 ) or CH2C(R 14 )(R 15 )(R 16 ). R 13 , R 14 , R 15 , R 16 , R’, R” and R sub are as defined above. In certain embodiments, R 13 is in each case independently H, C1-6 alkyl, cycloalkyl, aryl, heteroaryl or aralkyl. In certain embodiments, R 14 , R 15 and R 16 are each independently H, halo, C1-6 alkyl, alkaryl, aryl or heteroaryl. In certain embodiments, R’ and R” are each independently H, C1-6 alkyl, aryl or heteroaryl. In certain embodiments, one of R2 and R3 is H and the other is CN, CF3, CH2CF3, CF2H, CF2-phenyl, S(O)OR 13 , C(O)OR 13 , CH2S(O)OR 13 , CH2C(O)OR 13 or CONHR 13is an electron-withdrawing group such as. In certain embodiments, both R2 and R3 are CN, CF3, CH2CF3, CF2H, CF2-phenyl, S(O)OR 13 , C(O)OR 13 , CH2S(O)OR 13 , CH2C(O)OR 13 or CONHR 13 and the like are electron-withdrawing groups. In certain embodiments, R 13 is independently H, C1-3 alkyl, phenyl in each case.

[0014] In certain embodiments, [the cyclic disulfide moiety] has one of the following structures: [Chemical formula]

[0015] In certain embodiments, [the cyclic disulfide moiety] is [Chemical formula] (where n is 1 to 4). In certain embodiments, n is 2, 3 or 4. R2 and R3 are as defined above. In certain embodiments, R2 and R3 are each independently H, C1-6 alkyl, aryl, heteroaryl, CN or CH2CN, OR 13 or CH2OR 13 , C(O)OR 13 or CH2C(O)OR 13 , S(O)OR 13 or CH2S(O)OR 13 , C(O)N(R’)(R”) or CH2C(O)N(R’)(R”) or C(R 14 )(R 15 )(R 16 ) or CH2C(R 14 )(R 15 )(R 16 ) and each of them may optionally be substituted with one or more R sub groups. R 13 , R 14 , R 15 , R16 、R’, R” and R sub are as defined above. In certain embodiments, R 13 is, in each case independently, H, C1-6 alkyl, cycloalkyl, aryl, heteroaryl or aralkyl. In certain embodiments, R 14 , R 15 and R 16 are each independently H, halo, C1-6 alkyl, alkaryl, aryl or heteroaryl. In certain embodiments, R’ and R” are each independently H, C1-6 alkyl, aryl or heteroaryl. In certain embodiments, one of R2 and R3 is H and the other is alkyl, aryl, CF3, CH2CF3, CF2H, CF2-phenyl, S(O)OR 13 , C(O)OR 13 , CH2S(O)OR 13 , CH2C(O)OR 13 or CONHR 13 . In certain embodiments, both of R2 and R3 are H, alkyl, aryl, CF3, CH2CF3, CF2H, CF2-phenyl, S(O)OR 13 , C(O)OR 13 , CH2S(O)OR 13 , CH2C(O)OR 13 or CONHR 13 . In certain embodiments, R 13 is, in each case independently, H, C1-3 alkyl, phenyl.

[0016] In certain embodiments, [the cyclic disulfide moiety] has one of the following structures: [Chemical formula]

[0017] In certain embodiments, [the cyclic disulfide moiety] contains a disulfide-containing bridge, a bicyclic structure, i.e., in formula (C-I), (C-IIa) or (C-IIb), R3 and R5 together with the adjacent carbon atoms and two sulfur atoms form a second ring.

[0018] In certain embodiments, the [cyclic disulfide moiety] is

Chemical formula

[0019] In certain embodiments, [cyclic disulfide moiety] is:

Chemical formula

[0020] In certain embodiments, [cyclic disulfide moiety] has one of the following structures:

Chemical formula

[0021] In certain embodiments, [linker coupling group] is

Chemical formula

Chemical formula

[0022] In certain embodiments, the [linker coupling group] is [Chemical formula] has the structure of. In this formula, X1 and Z1 are each independently OH, OM, OR 13 , SH, SM, SR 13, C(O)H, S(O)H, C1-C6 alkyl optionally substituted by one or more hydroxy or halo groups, NSO2R’, N(R’)(R”), N=CN(R’)(R”), or D-Q; D is independently absent, O, S, NH, C1-C6 alkylene optionally substituted by one or more halo groups in each case; and Y1 is S or O. In certain embodiments, X1 is OH or SH; and Z1 is D-Q.

[0023] In certain embodiments, [linker coupling group] has one of the following structures:

Chemical formula

Chemical formula

Chemical formula

[0024] In certain embodiments, [linker coupling group] is

Chemical formula

[0025] In certain embodiments, [linker coupling group] is

Chemical formula

[0026] In one embodiment, the compound has one of the following structures:

Table 1

[0027] In one embodiment, the compound has one of the following structures:

Table 2

Table 3

Table 4

Table 5

[0028] In one embodiment, the compound has one of the following structures:

Table 6

Table 7

Table 8

[0029] In one embodiment, the compound comprises a stereoisomer of formula (I) having a chiral purity of at least 70%.

[0030] In one embodiment, the compound comprises one of the following stereoisomers having a chiral purity of at least 70%:

Table 9

[0031] In one embodiment, the compound comprises the following stereoisomer having a chiral purity of at least 70%:

Table 10

[0032] In certain embodiments, the [linker coupling group] has the structure (P-I), and [cyclic disulfide moiety]-P(Y1)(X1)- is

Chemical Formula

[0033] In certain embodiments, the compound optionally comprises one or more ligands connected via one or more linkers to any of R2, R3, R4, R5, R6, R7, R8 and R9 of the [cyclic disulfide moiety].

[0034] In certain 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, a folate, a thyrotropin, a melanotropin, surfactant protein A, a mucin, a glycosylated polyamino acid, a transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, 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.

[0035] In certain embodiments, at least one ligand is a carbohydrate-based ligand that targets liver tissue. In certain embodiments, the carbohydrate-based ligand is selected from the group consisting of galactose, polyvalent galactose, N-acetyl-galactosamine (GalNAc), polyvalent GalNAc, mannose, polyvalent mannose, lactose, polyvalent lactose, N-acetyl-glucosamine (GlcNAc), polyvalent GlcNAc, glucose, polyvalent glucose, fucose, and polyvalent fucose.

[0036] In certain embodiments, the carbohydrate-based ligand is an ASGPR ligand. For example, the ASGPR ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker, such as [Chemical formula] .

[0037] In certain embodiments, at least one ligand is a lipophilic moiety. In certain embodiments, the lipophilicity of the lipophilic moiety is greater than 0 as measured by logK ow or the hydrophobicity of the compound is greater than 0.2 as measured by the unbound fraction in the plasma protein binding assay of the compound.

[0038] In certain embodiments, the lipophilic moiety comprises a saturated or unsaturated C4-C 30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety comprises a saturated or unsaturated C6-C 18 hydrocarbon chain.

[0039] In certain embodiments, at least one ligand targets a receptor involved in delivery to the CNS tissue. In certain 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.

[0040] In certain embodiments, at least one ligand targets a receptor involved in delivery to the eye tissue. In certain embodiments, the ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate-based ligand.

[0041] Another aspect of the present invention relates to an oligonucleotide (e.g., a single-stranded iRNA agent or a double-stranded iRNA agent) comprising one or more structures of formula (I): [Cyclic disulfide moiety]-[Linker coupling group] (I).

[0042] Another aspect of the present invention relates to an oligonucleotide (e.g., a single-stranded iRNA agent or a double-stranded iRNA agent) comprising one or more structures of formula (II): [Cyclic disulfide moiety]-P(Y)(X)- * (II).

[0043] In both formula (I) and (II), the [Cyclic disulfide moiety] is

Chemical formula

Chemical formula

[0044] In formula (I), at least one [linker coupling group] contains a nucleoside or oligonucleotide.

[0045] In formula (II), * represents a bond to an oligonucleotide, Y is absent, N(R’), =O or =S; X is OH, SH, C(O)H, S(O)H, optionally one or more R sub alkyl substituted with groups, N(R’)(R”), NSO2R’, N=CN(R’)(R”), B(R 13 )3, BH3 - or X’, where X’ is N(R’)(R”), OR 13 or SR 13 is.

[0046] All of the above embodiments regarding formula (C-I) and formula (C-IIa) (or C-IIb) of [cyclic disulfide moiety], all of the formulas of [linker coupling group], all of the variable groups defined by these formulas, all ligands and all subgenera and species structures related to this compound, the [cyclic disulfide moiety] and [linker coupling group] in the first aspect of the present invention related to this compound are suitable in these aspects of the present invention to which the oligonucleotide is related.

[0047] In certain embodiments, the [cyclic disulfide moiety] has a structure selected from the following groups Ia), Ib) and II). Group Ia) includes the following structures:

Chemical formula

Chemical formula

Chemical formula

[0048] In certain embodiments, the [cyclic disulfide moiety] has a structure selected from one of the structures from group III). Group III) includes the following structures:

Chemical formula

[0049] In certain embodiments, the oligonucleotide comprises a structure selected from the group consisting of the following

Chemical formula

[0050] In certain embodiments, the oligonucleotide comprises an enantiomer of formula (I) or (II) having a chiral purity of at least 70%.

[0051] In certain embodiments, the oligonucleotide comprises the following enantiomer having a chiral purity of at least 70%.

Table 11

[0052] In certain embodiments, the compound comprises the following enantiomer having a chiral purity of at least 70%.

Table 12

[0053] In certain embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(O)(SH)- * or a salt thereof.

[0054] In certain embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(O)(OH)- * or a salt thereof.

[0055] In certain embodiments, the oligonucleotide has the formula: [Cyclic disulfide moiety]-P(O)(OR13)- * or a salt thereof. The variable group R 13 is as defined above.

[0056] In certain embodiments, the oligonucleotide has a structure of the formula: [Cyclic disulfide moiety]-P(S)(OR13)- * or a salt thereof. The variable group R 13 is as defined above.

[0057] In certain embodiments, the oligonucleotide has a structure of the formula: [Cyclic disulfide moiety]-P(O)R13- * or a salt thereof. The variable group R 13 is as defined above.

[0058] In certain embodiments, the oligonucleotide has a structure of the formula: [Cyclic disulfide moiety]-P(S)(SH)- * or a salt thereof.

[0059] In certain embodiments, the oligonucleotide has a structure of the formula: [Cyclic disulfide moiety]-P(O)N(R’)(R”)- * or a salt thereof. The variable groups R’ and R” are as defined above.

[0060] In certain embodiments, the oligonucleotide has a structure of the formula: [Cyclic disulfide moiety]-P(O)NSO2R’- * or a salt thereof. The variable group R’ is as defined above.

[0061] In certain embodiments, the oligonucleotide has a structure of the formula: [Cyclic disulfide moiety]-P(O)N=CN(R’)(R”))- * or a salt thereof. The variable groups R’ and R” are as defined above.

[0062] In certain embodiments, the oligonucleotide comprises a structure having one of the following formulas:

Chemical formula

[0063] In certain embodiments, the [cyclic disulfide moiety] has one of the following structures: [Table 13] Here, * is -P(X)(Y)- * represents the bond to the phosphorus atom of the group.

[0064] In certain embodiments, the compound has one of the following structures: [Table 14]

[0065] In certain embodiments, the oligonucleotide [Chemical formula] contains a structure selected from the group consisting of. X is O or S.

[0066] In certain embodiments, the oligonucleotide contains at least one [cyclic disulfide moiety] at the 5'-end of the oligonucleotide.

[0067] In certain embodiments, the first nucleotide at the 5'-end of the oligonucleotide is [Chemical formula] has the structure of or a salt thereof, where: R S is the [cyclic disulfide moiety]; X is -OH, -SH, C(O)H, S(O)H, optionally alkyl substituted with one or more R sub groups, N(R')(R"), NSO2R', N=CN(R')(R"), B(R 13 )3, BH3 - or X', where X' is N(R')(R"), -OR 13 or -SR 13 ; Y is S, O or N(R'); Z is O, S, N(R’), or CH2; and [Modified sugar] is a sugar moiety containing one or more sugar modifications selected from the group consisting of 2’-modification, LNA, isomer modification, 5’-modification, non-natural cyclic modification, acyclic modification, and abasic modification.

[0068] In certain embodiments, the sugar modification in [modified sugar] is 2’-modification, LNA, isomer modification, 5’-modification, or abasic modification.

[0069] In certain embodiments, the first nucleotide at the 5’-end of the oligonucleotide is

Chemical formula

[0070] In these formulas, * represents a bond to the following optionally modified internucleotide linkage; B is an optionally modified nucleobase or H; R S is [cyclic disulfide moiety]; and R1 is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH2-allyl, fluoro, O-N-methylacetamide (O-NMA), O-N-alkylacetamide, O-dimethoxypropyl, O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP), or ara-F; or R1 forms a bridge with the 4’-carbon; R2 is H, alkyl, or aryl; X is -OH, -SH, C(O)H, S(O)H, alkyl optionally substituted with one or more R sub groups, N(R’)(R”), NSO2R’, N=CN(R’)(R”), B(R 13 )3, BH3 - or X’, where X’ is N(R’)(R”), -OR 13 or -SR 13 ; R 13 is independently in each case H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl or arylcarbonyl, each of which may optionally be substituted with one or more R sub groups; R sub is independently in each case halo, haloalkyl, alkyl, alkaryl, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, arylcarbonyl, acyloxy, cyano or ureido; Y is S, O or N(R’); Z is O, S, N(R’) or CH2; and Q is O, S, CH2 or N(R’).

[0071] In certain embodiments, the sugar modification in [modified sugar] is a 2’ modification. In certain embodiments, the first nucleotide at the 5’ end of the oligonucleotide has one of the following structures:

Chem.

[0072] In certain embodiments, the sugar modification in [modified sugar] is a "locked" nucleic acid (LNA). In certain embodiments, the first nucleotide at the 5’ end of the oligonucleotide is:

Chem.

[0073] In certain embodiments, the sugar modification in [modified sugar] is an isomeric modification to ribose sugar. In certain embodiments, the first nucleotide at the 5' end of the oligonucleotide has one of the following structures:

Chemical formula

[0074] In certain embodiments, the sugar modification in [modified sugar] is a 5'-modification having a substituent at the 5'-position of ribose sugar. In certain embodiments, the first nucleotide at the 5' end of the oligonucleotide has one of the following structures:

Chemical formula

[0075] In certain embodiments, the sugar modification in [modified sugar] is a deoxybase modification. In certain embodiments, the first nucleotide at the 5' end of the oligonucleotide has one of the following structures:

Chemical formula

[0076] In certain embodiments, [modified sugar] includes a non-natural cyclic modification having one of the following structures:

Chemical formula

[0077] In certain embodiments, the first nucleotide at the 5' end of the oligonucleotide has one of the following structures:

Chemical formula

[0078] In certain embodiments, [modified sugar] includes an acyclic modification having one of the following structures:

Chemical formula

[0079] In certain embodiments, the first nucleotide at the 5' end of the oligonucleotide has one of the following structures:

Chemical formula

[0080] In certain embodiments, the first nucleotide at the 5' end of the oligonucleotide has one of the following structures (

Chemical formula

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

[0081] In certain embodiments, the first nucleotide at the 5'-end of the oligonucleotide has the structure:

Chemical formula

[0082] In certain embodiments, the first nucleotide at the 5'-end of the oligonucleotide has the structure:

Chemical formula

[0083] In certain embodiments, the first nucleotide at the 5'-end of the oligonucleotide has the structure:

Chemical formula

[0084] In certain embodiments, B or Base in all of these sugars or modified sugar structures described above is uridine. In certain embodiments, R or R1 in all of these sugars or modified sugar structures described above is hydroxy or methoxy. In certain embodiments, R or R1 in all of these sugars or modified sugar structures described above is hydrogen.

[0085] In certain embodiments, the oligonucleotide comprises at least one [cyclic disulfide moiety] at the 3'-terminus of the oligonucleotide.

[0086] In certain embodiments, the oligonucleotide comprises at least one [cyclic disulfide moiety] at the 5'-terminus of the oligonucleotide.

[0087] In certain embodiments, the oligonucleotide comprises at least one [cyclic disulfide moiety] at the 5'-terminus of the oligonucleotide and at least one [cyclic disulfide moiety] at the 3'-terminus of the oligonucleotide.

[0088] In certain embodiments, the oligonucleotide comprises at least one [cyclic disulfide moiety] at an internal position of the oligonucleotide.

[0089] In certain embodiments, the oligonucleotide is a single-stranded oligonucleotide.

[0090] In certain embodiments, the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand.

[0091] In certain embodiments, the sense and antisense strands are each 15 to 30 nucleotides in length. In certain embodiments, the sense and antisense strands are each 19 to 25 nucleotides in length. In certain embodiments, the sense and antisense strands are each 21 to 23 nucleotides in length.

[0092] In certain embodiments, the oligonucleotide comprises a single-stranded overhang at at least one end, e.g., a 3' and / or 5' overhang that is 1 to 10 nucleotides in length, e.g., an overhang that is 1, 2, 3, 4, 5, or 6 nucleotides in length. In certain 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 certain embodiments, the single-stranded overhang is, optionally, 1, 2, or 3 nucleotides in length at at least one end.

[0093] In certain embodiments, the oligonucleotide may also have blunt ends located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. In certain 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 certain 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 certain embodiments, the oligonucleotide has two blunt ends at both ends of the double-stranded iRNA duplex.

[0094] In certain embodiments, the sense strand of the oligonucleotide is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where these strands form a double-stranded region of 21 contiguous base pairs having a 2-nucleotide-long single-stranded overhang at the 3' end.

[0095] In certain embodiments, the sense strand comprises at least one [cyclic disulfide moiety]. In certain embodiments, the antisense strand comprises at least one [cyclic disulfide moiety]. In certain embodiments, both the sense strand and the antisense strand each comprise at least one [cyclic disulfide moiety].

[0096] 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.

[0097] In certain embodiments, the sense strand further comprises at least one phosphorothioate bond at the 3' end. In certain embodiments, the sense strand comprises at least two phosphorothioate bonds at the 3' end.

[0098] In certain embodiments, the sense strand further comprises at least one phosphorothioate bond at the 5' end. In certain embodiments, the sense strand comprises at least two phosphorothioate bonds at the 5' end.

[0099] In certain embodiments, the antisense strand further comprises at least one phosphorothioate bond at the 3' end. In certain embodiments, the antisense strand comprises at least two phosphorothioate bonds at the 3' end.

[0100] 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).

[0101] In certain embodiments, the 5' end of the antisense strand does not comprise 5'-vinylphosphonate (VP).

[0102] In certain embodiments, the oligonucleotide further comprises at least one terminal chiral phosphorus atom.

[0103] Site-specific, chiral modification of the internucleotide linkage occurs at the 5' end, 3' end, or both the 5' and 3' ends of the strand. This is herein referred to as "terminal" chiral modification. Terminal modification can occur at the 3' or 5' terminal position of the terminal region, for example, at the position of the terminal nucleotide of the strand or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Chiral modification can occur on the sense strand, the antisense strand, or both the sense and antisense strands. Each of the chirally pure phosphorus atoms can be in the Rp configuration, the Sp configuration, or combinations thereof. Details of the chiral modification and the chirally modified dsRNA agents can be found in PCT / US18 / 67103, entitled "Chirally-Modified Double-Stranded RNA Agents," filed on December 21, 2018, which is hereby incorporated by reference in its entirety.

[0104] In certain embodiments, the oligonucleotide further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3' end of the antisense strand having a 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 having a 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 having a linking phosphorus atom in the Rp or Sp configuration.

[0105] In certain embodiments, the oligonucleotide further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3' end of the antisense strand having a 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 having a 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 having a linking phosphorus atom in either the Rp or Sp configuration.

[0106] In certain embodiments, the oligonucleotide further comprises chiral modifications at the termini resulting from the internucleotide linkages between the first, second, and third nucleotides at the 3’-end of the antisense strand having a phosphorus atom in the Sp configuration; chiral modifications at the termini resulting from the internucleotide linkage of the first nucleotide at the 5’-end of the antisense strand having a phosphorus atom in the Rp configuration; and chiral modifications at the termini resulting from the internucleotide linkage of the first nucleotide at the 5’-end of the sense strand having a phosphorus atom in either the Rp or Sp configuration.

[0107] In certain embodiments, the oligonucleotide further comprises chiral modifications at the termini resulting from the internucleotide linkages between the first and second nucleotides at the 3’-end of the antisense strand having a phosphorus atom in the Sp configuration; chiral modifications at the termini resulting from the internucleotide linkage of the third nucleotide at the 3’-end of the antisense strand having a phosphorus atom in the Rp configuration; chiral modifications at the termini resulting from the internucleotide linkage of the first nucleotide at the 5’-end of the antisense strand having a phosphorus atom in the Rp configuration; and chiral modifications at the termini resulting from the internucleotide linkage of the first nucleotide at the 5’-end of the sense strand having a phosphorus atom in either the Rp or Sp configuration.

[0108] In certain embodiments, the oligonucleotide further comprises chiral modifications at the termini resulting from the internucleotide linkages between the first and second nucleotides at the 3’-end of the antisense strand having a phosphorus atom in the Sp configuration; chiral modifications at the termini resulting from the internucleotide linkages between the first and second nucleotides at the 5’-end of the antisense strand having a phosphorus atom in the Rp configuration; and chiral modifications at the termini resulting from the internucleotide linkage of the first nucleotide at the 5’-end of the sense strand having a phosphorus atom in either the Rp or Sp configuration.

[0109] In certain embodiments, the oligonucleotide has at least two phosphorothioate internucleotide linkages in the first 5 nucleotides of the antisense strand (counting from the 5’-end).

[0110] In certain embodiments, the antisense strand comprises two blocks of phosphorothioate nucleotide internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate nucleotide internucleotide linkages.

[0111] In certain embodiments, the oligonucleotide comprises one or more targeting ligands that are attached, optionally via one or more linkers, to any of R2, R3, R4, R5, R6, R7, R8, and R9 of the [cyclic disulfide moiety] of the compound.

[0112] In certain 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, a folate, a thyrotropin, a melanotropin, surfactant protein A, a mucin, a glycosylated polyamino acid, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipophilic moiety that enhances plasma protein binding, cholesterol, a steroid, a bile acid, vitamin B12, biotin, a fluorophore, and a peptide.

[0113] In certain embodiments, at least one targeting ligand is a lipophilic moiety. In certain embodiments, the lipophilicity of the lipophilic moiety is measured by logK ow and is greater than 0 or the hydrophobicity of the compound is measured by the unbound fraction in a plasma protein binding assay of the compound and is greater than 0.2. In certain embodiments, the lipophilic moiety comprises a saturated or unsaturated C4-C 30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety comprises a saturated or unsaturated C6-C 18 hydrocarbon chain.

[0114] In certain embodiments, at least one targeting ligand targets a receptor involved in delivery to a particular CNS tissue. In certain 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.

[0115] In certain embodiments, at least one targeting ligand targets a receptor involved in delivery to eye tissue. In certain embodiments, the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate-based ligand. In certain embodiments, the targeting ligand is an RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO: 328) or cyclo(-Arg-Gly-Asp-D-Phe-Cys) (SEQ ID NO: 329).

[0116] In certain embodiments, at least one targeting ligand targets liver tissue. In certain embodiments, the targeting ligand is a carbohydrate-based ligand. In certain embodiments, the carbohydrate-based ligand is selected from the group consisting of galactose, polyvalent galactose, N-acetyl-galactosamine (GalNAc), polyvalent GalNAc, mannose, polyvalent mannose, lactose, polyvalent lactose, N-acetyl-glucosamine (GlcNAc), polyvalent GlcNAc, glucose, polyvalent glucose, fucose, and polyvalent fucose. In certain embodiments, the targeting ligand is a GalNAc conjugate. For example, the GalNAc conjugate is one or more GalNAc derivatives linked via a divalent or trivalent branched linker, such as:

Chemical formula

[0117] In certain 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 all the nucleotides present in the oligonucleotide contain the modifications described herein.

[0118] In certain embodiments, the antisense and sense strands of the oligonucleotide contain at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or virtually 100% 2'-O-methyl modified nucleotides.

[0119] 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.

[0120] In certain embodiments, the oligonucleotide is antisense, and at least 50% of the antisense nucleotides are independently modified with LNA, CeNA, 2'-methoxyethyl or 2'-deoxy.

[0121] In certain embodiments, the sense and antisense strands contain 12 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or fewer 2'-F modified nucleotides or do not contain any. In certain embodiments, the oligonucleotide has 12 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or fewer 2'-F modifications in the sense strand or does not have any. In certain embodiments, the oligonucleotide has 12 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or fewer 2'-F modifications in the antisense strand or does not have any. In certain embodiments, the sense and antisense strands contain 10 or fewer 2'-fluoro modified nucleotides.

[0122] 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'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP) and 2'-ara-F.

[0123] In certain embodiments, the oligonucleotide comprises one or more 2'-F modifications at any position of the sense or antisense strand.

[0124] In certain 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-methoxypropyl), 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (e.g., 2'-modified L-nucleosides, e.g., 2'-deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic and acyclic alkyls.

[0125] In certain embodiments, the oligonucleotide has greater than 80%, greater than 85%, greater than 90%, greater than 95% or virtually 100% natural nucleotides. For the purposes of these embodiments, natural nucleotides can include those having 2'-OH, 2'-deoxy and 2'-OMe.

[0126] In certain embodiments, the antisense strand comprises, for example, at least one unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA) modification in the seed region of the antisense strand. In certain embodiments, the seed region is positions 2-8 (or 5-7) of the 5' end of the antisense strand.

[0127] In certain embodiments, the oligonucleotides each have a sense strand and an antisense strand that are 15-30 nucleotides in length; the antisense strand contains at least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides; wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the oligonucleotides have less than 20%, less than 15%, less than 10%, less than 5% unnatural nucleotides or are substantially free of unnatural nucleotides.

[0128] In certain embodiments, the oligonucleotides each have a sense strand and an antisense strand that are 15-30 nucleotides in length; the antisense strand contains at least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides; wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the oligonucleotides have greater than 80%, greater than 85%, greater than 95%, or virtually 100% natural nucleotides, such as those having 2'-OH, 2'-deoxy, or 2'-OMe.

[0129] Certain aspects of the invention provide oligonucleotides each independently having a sense strand and an antisense strand that are 15-35 nucleotides in length; at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the antisense strand; the sense and / or antisense strands comprising at least 3, 4, 5, or 6 2'-deoxy modifications; wherein the oligonucleotides have a double-stranded (duplex) region of 19-25 base pairs; and wherein the oligonucleotides contain a ligand.

[0130] In certain embodiments, the sense strand does not contain glycol nucleic acid (GNA).

[0131] It is understood that the antisense strand has sufficient complementarity to the target sequence for the mediation of RNA interference. In other words, the oligonucleotide can inhibit the expression of the target gene.

[0132] In certain embodiments, the oligonucleotide comprises at least three 2'-deoxy modifications. The 2'-deoxy modifications are at the 2nd and 14th positions of the antisense strand when counted from the 5' end of the antisense strand and at the 11th position of the sense strand when counted from the 5' end of the sense strand.

[0133] In certain embodiments, the oligonucleotide comprises at least five 2'-deoxy modifications. The 2'-deoxy modifications are at the 2nd, 12th, and 14th positions of the antisense strand when counted from the 5' end of the antisense strand and at the 9th and 11th positions of the sense strand when counted from the 5' end of the sense strand.

[0134] In certain embodiments, the oligonucleotide comprises at least seven 2'-deoxy modifications. The 2'-deoxy modifications are at the 2nd, 5th, 7th, 12th, and 14th positions of the antisense strand when counted from the 5' end of the antisense strand and at the 9th and 11th positions of the sense strand when counted from the 5' end of the sense strand.

[0135] In certain embodiments, the antisense strand comprises at least five 2'-deoxy modifications at the 2nd, 5th, 7th, 12th, and 14th positions when counted from the 5' end of the antisense strand. The antisense strand has a length of 18 - 25 nucleotides or 18 - 23 nucleotides.

[0136] In certain embodiments, the oligonucleotide contains less than 20%, for example, less than 15%, less than 10%, or less than 5% non-natural nucleotides or does not contain non-natural nucleotides.

[0137] In certain embodiments, the sense strand does not contain glycol nucleic acid (GNA); where the oligonucleotide contains less than 20%, for example, less than 15%, less than 10% or less than 5% of non-natural nucleotides or contains all natural nucleotides.

[0138] In certain embodiments, at least one sense strand and antisense strand contain at least one, for example, at least two, at least two, 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 or antisense strand.

[0139] In certain embodiments, the sense strand and / or antisense strand contain at least one, for example, at least two, at least two, 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.

[0140] In certain embodiments, the sense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the sense strand. For example, the sense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications within positions 7, 8, 9, 10, 11, 12 and 13 counted from the 5' end of the sense strand.

[0141] In certain embodiments, the antisense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications in the central region of the antisense strand. For example, the antisense strand has a length of 18 to 30 nucleotides and contains at least two 2'-deoxy modifications within positions 10, 11, 12, 13, 14, 15 and 16 counted from the 5' end of the antisense strand.

[0142] In certain embodiments, the oligonucleotide comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17 to 30 nucleotides and comprises at least one 2'-deoxy modification in the central region of the sense strand; wherein the antisense strand independently has a length of 17 to 30 nucleotides and comprises at least two 2'-deoxy modifications in the central region of the antisense strand.

[0143] In certain embodiments, the oligonucleotide comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17 to 30 nucleotides and comprises at least two 2'-deoxy modifications in the central region of the sense strand; wherein the antisense strand independently has a length of 17 to 30 nucleotides and comprises at least one 2'-deoxy modification in the central region of the antisense strand.

[0144] In certain embodiments, the sense strand comprises at least one, for example, 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.

[0145] In certain embodiments, the antisense strand comprises at least one, for example, 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 antisense strand.

[0146] In certain embodiments, the oligonucleotide comprises less than 20%, for example, less than 15%, less than 10% or less than 5% non-natural nucleotides or the oligonucleotide comprises all natural nucleotides; wherein the sense strand and / or the antisense strand comprises at least one, for example, 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 the antisense strand.

[0147] In certain embodiments, the oligonucleotide comprises less than 20%, such as less than 15%, less than 10% or less than 5% non-natural nucleotides or the oligonucleotide comprises all natural nucleotides; wherein the sense strand comprises at least one, such as 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.

[0148] In certain embodiments, the oligonucleotide comprises less than 20%, such as less than 15%, less than 10% or less than 5% non-natural nucleotides or the oligonucleotide comprises all natural nucleotides; wherein the antisense strand comprises at least one, such as 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 antisense strand.

[0149] In certain embodiments, when the oligonucleotide comprises less than 8 non-2'OMe nucleotides, the antisense stand comprises at least one DNA. For example, in any of the embodiments of the present invention, when the oligonucleotide comprises less than 8 non-2'OMe nucleotides, the antisense stand comprises at least one DNA.

[0150] In certain embodiments, when the antisense comprises two deoxynucleotides which are at the 2nd and 14th positions counted from the 5'-end of the antisense strand, the oligonucleotide comprises 8 or less (such as 8, 7, 6, 5, 4, 3, 2, 1 or 0) non-2'OMe nucleotides. For example, in any of the embodiments of the present invention, when the antisense comprises two deoxynucleotides which are at the 2nd and 14th positions counted from the 5'-end of the antisense strand, the oligonucleotide comprises 0, 1, 2, 3, 4, 5, 6, 7 or 8 non-2'-OMe nucleotides.

[0151] Another aspect of the invention relates to a pharmaceutical composition comprising the oligonucleotides described herein and pharmaceutically acceptable additives.

[0152] All of the above embodiments related to the oligonucleotides in the above aspect of the invention related to oligonucleotides are applicable to this aspect of the invention related to the pharmaceutical composition.

[0153] In another aspect, the invention further provides a method for delivering the oligonucleotides of the invention to a specific target in a subject rather than by subcutaneous or intravenous administration. The invention further provides the oligonucleotides of the invention for use in a method for delivering the agent to a specific target in a subject by subcutaneous or intravenous administration.

[0154] Another aspect of the invention relates to a method for reducing or inhibiting the expression of a target gene in a subject, the method comprising administering to the subject an amount of the oligonucleotide sufficient to inhibit the expression of the target gene.

[0155] All of the above embodiments related to the oligonucleotides in the above aspect of the invention related to oligonucleotides are applicable to this aspect of the invention related to a method for reducing or inhibiting the expression of a target gene in a subject.

[0156] Another aspect of the invention relates to a method for modifying an oligonucleotide, the method comprising contacting the oligonucleotide with the above compound under conditions suitable for the reaction of the compound with the oligonucleotide, wherein the oligonucleotide contains a free hydroxyl group.

[0157] 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.

[0158] In certain embodiments, the oligonucleotide contains a 5'-OH group. In certain embodiments, the oligonucleotide contains a 3'-OH group.

[0159] In one embodiment, the conditions suitable for the reaction of the compound and the oligonucleotide are a medium containing an acid catalyst. For example, the acid catalyst can be a substituted tetrazole.Suitable acidic catalysts include, but are not limited to, 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-hydroxylbenzotriazole, 1-hydroxy-6-trifluoromethylbenzotriazole, 4-nitro-1-hydroxy-6-trifluoromethylbenzotriazole, pyridinium chloride, pyridinium bromide, 4-methylbenzenesulfonic acid pyridinium, 2,6-di(tert-butyl)pyridinium chloride, pyridinium trifluoroacetate, N-(phenyl)imidazolium triflate (N-PhIMT), N-(phenyl)-imidazolium perchlorate (N-PhIMP), N-(methyl)benzimidazolium triflate (NMeBIT), N-(p-acetylphenyl)imidazolium triflate (N-AcPhIMT), N-(phenyl)imidazolium tetrafluoroborate (N-PhIMTFB), imidazolium perchlorate (IMP), 4-(phenyl)-imidazolium triflate (4-PhIMT), benzimidazolium tetrafluoroborate (BITFB), imidazolium tetrafluoroborate (IMTFB), imidazolium triflate (IMT), benzimidazolium triflate (BIT), 2-(phenyl)imidazolium triflate (2-PhIMT), N-(methyl)imidazolium triflate (N-MeIMT), 4-(methyl)imidazolium 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(III) chloride (FeCl3), aluminum chloride (AlCl3), boron trifluoride etherate (BF3-OEt2), zirconium(IV) chloride (ZrCl4) and bismuth(III) chloride (BiCl3), trimethylchlorosilane, 2,4-dinitrophenol, 1-methyl-5-mercapto-tetrazole and 1-phenyl-5-mercaptotetrazole.

[0160] All of the above embodiments related to the compounds and oligonucleotides in the above aspects of the present invention are suitable for this aspect of the present invention related to methods of modifying oligonucleotides.

[0161] Another aspect of the present invention is a method for producing a modified oligonucleotide, of formula (A):

Chemical formula

Chemical formula

[0162] In certain 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 certain 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).

[0163] In certain embodiments, the first nucleotide at the 5' end of the first oligonucleotide is of formula (C):

Chemical formula

[0164] In certain embodiments, the first nucleotide at the 5' end of the modified oligonucleotide is of formula (D):

Chemical formula

[0165] In certain embodiments, the first nucleotide at the 5' end of the modified oligonucleotide is of formula (E) or (F):

Chemical formula

[0166] In certain embodiments, the conditions suitable for forming the modified oligonucleotide include the use of 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-dithiazole-5-thione (DDTT); dimethyl sulfoxide; and N-bromosuccinimide. For example, the oxidizing agent can be a peracid (e.g., m-chloroperbenzoic acid) or a peroxide (e.g., tert-butyl hydroperoxide or trimethylsilyl peroxide).

[0167] All of the above embodiments related to the compounds and oligonucleotides in the above-described embodiments of the present invention are applicable to this aspect of the present invention related to a method for producing a modified oligonucleotide.

[0168] Another aspect of the present invention is a precursor formula (I): [Cyclic disulfide moiety]-[Linker coupling group] (I) [Wherein, [Cyclic disulfide moiety] is

Chemical formula

[0169] In certain embodiments, [the cyclic disulfide moiety] is

Chemical formula

[0170] In certain embodiments, [the cyclic disulfide moiety] is

Chemical formula

[0171] In certain embodiments, [the cyclic disulfide moiety] is

Chemical formula

[0172] In certain embodiments, [the cyclic disulfide moiety] is

Chemical formula

[0173] In certain embodiments, W in R2 is NHTFA (CF3C(O)N(H)-), N3, C≡CH, C(O)OR 13 or OC(O)R 13 where R 13 is C1-C3 alkyl.

[0174] In certain embodiments, [the cyclic disulfide moiety] has one of the following structures:

Chemical formula

[0175] In certain embodiments, [the linker coupling group] is

Chemical formula

[0176] In certain embodiments, [linker coupling group] is

Chemical formula

[0177] In certain embodiments, [linker coupling group] is

Chemical formula

[0178] In certain embodiments, [linker coupling group] is

Chemical formula

[0179] In certain embodiments, the compound has one of the following structures:

Table 22

[0180] Another aspect of the present invention includes one or more structures of formula (II) or salts or stereoisomers thereof: [Cyclic disulfide moiety]-P(Y)(X)- * (II) 〔wherein, [Cyclic disulfide moiety] is

Chemical formula

[0181] In certain embodiments, the [cyclic disulfide moiety] is [Chemical formula] has the structure of. R2 is as defined above. R4 and R5 are each independently H, C1-6 alkyl or phenyl. Alternatively, R4 and R5 together with the adjacent carbon atoms form a second ring of 3 to 7 atoms.

[0182] In certain embodiments, the [cyclic disulfide moiety] is [Chemical formula] has the structure of.

[0183] [Cyclic disulfide moiety] is [Chemical formula] has the structure of. R2 is as defined above. R4 and R5 are each independently H, C1-6 alkyl or phenyl. Alternatively, R4 and R5, together with the adjacent carbon atoms, form a second ring of 3 to 7 atoms.

[0184] In certain embodiments, [cyclic disulfide moiety] is [Chemical formula] has the structure of.

[0185] In certain embodiments, W in R2 is NHTFA, N3, C≡CH, C(O)OR 13 , OC(O)R 13 , where R 13 is C1-C3 alkyl.

[0186] In certain embodiments, [cyclic disulfide moiety] has one of the following structures: [Chemical formula] [wherein, TFAHN- is CF3C(O)N(H)-].

[0187] In certain embodiments, the oligonucleotide is of the formula: [cyclic disulfide moiety]-P(O)(SH)- * , [cyclic disulfide moiety]-P(O)(OH)- * , [cyclic disulfide moiety]-P(O)(OR 13 )- * , [cyclic disulfide moiety]-P(S)(OR 13 )- * , [cyclic disulfide moiety]-P(S)(SH)- *, [Cyclic disulfide moiety]-P(O)N(R’)(R”)- * , [Cyclic disulfide moiety]-P(O)NSO2R’- * , [Cyclic disulfide moiety]-P(O)N=CN(R’)(R”))- * , [Cyclic disulfide moiety]-P(O)R 13 - * comprises a structure having - or a salt thereof. R 13 , R’, R’’ and * are as defined above.

[0188] The precursor compounds and oligonucleotides containing the precursor compounds provided herein contain reactive groups for promoting 5'-end conjugation. For example, a [cyclic disulfide moiety] containing a reactive group W can react with a ligand containing a reactive functional group with a W-reactive group (NHTFA, N3, C≡CH, C(O)O-, OC(O), etc.) for further 5'-end conjugation. In certain embodiments, a [cyclic disulfide moiety] containing N3 or C≡CH can be reacted via click chemistry with a corresponding ligand containing C≡CH or N3, respectively.

Brief Description of the Drawings

[0189]

Figure 1

[0190]

Figure 2

[0191]

Figure 3

[0192]

Figures 4A-J

[0193]

Figure 5

[0194]

Figure 6

[0195]

Figure 7

[0196]

Figure 8

[0197]

Figure 9

[0198]

Figure 10

[0199]

Figure 11

[0200]

Figure 12

[0201]

Figure 13

[0202]

Figure 14

[0203]

Figures 15A-15B

[0204]

Figures 16A-16B

[0205]

Figures 17A-17B

[0206]

Figure 18

[0207]

Figure 19

[0208]

Figures 20A-20B

[0209]

Figure 21

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Figures 22A-22B

[0211]

Figure 23

[0212]

Figure 24

[0213]

Figures 25A-25B

[0214]

Figure 26

[0215]

Figure 27

Mode for Carrying Out the Invention

[0216] Detailed Description The inventors have discovered a new category of cyclic disulfide moieties that can be introduced into the phosphate groups of oligonucleotides (e.g., single-stranded iRNA agents and double-stranded iRNA agents) for temporarily masking phosphate groups and can be cleaved in vivo via cell activation. Cell activation occurs via a glutathione or dithiothreitol-mediated reduction / bio-conversion mechanism for releasing the phosphate group in the active anionic form from the masking group. The inventors have discovered that the cyclic disulfide moiety can be introduced at the 5' end, 3' end, and / or internal position of the sense strand or antisense strand or both the sense strand and antisense strand. The introduction of a cyclic disulfide moiety-modified phosphate prodrug at the 5' end of the antisense strand provides particularly good results.

[0217] Modified phosphate prodrug compounds One aspect of the present invention relates to a modified phosphate prodrug compound. The compound comprises a structure of formula (I): [Cyclic disulfide moiety]-[Linker coupling group] (I). The compound may also include a salt or stereoisomer of the structure of formula (I).

[0218] [Cyclic disulfide moiety] is

Chemical Formula

Chemical formula

[0219] In formulas (C-I), (C-IIa), (C-IIb) and (C-III): R1 is O or S and is bonded to the P atom of [the linker coupling group];

Chemical formula

[0220] In certain embodiments, in [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 may optionally be substituted with one or more R sub groups; 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 may optionally be substituted with one or more R sub groups; or R3 and R5 together with the adjacent carbon atoms and two sulfur atoms form a second ring of 6-8 atoms; R 13 is in each case independently H, C1-C6 alkyl, aryl, alkylcarbonyl or arylcarbonyl; and R’ and R” are each independently H or C1-C6 alkyl. R2, R4, R6, R7, R8 and R9 are each independently CN or C1-C6 alkylene-CN, C(O)OR 13 or C1-C6 alkylene-C(O)OR 13 , S(O)OR 13 or C1-C6 alkylene-S(O)OR 13 , C(O)N(R’)(R”) or C1-C6 alkylene-C(O)N(R’)(R”), each of which may optionally be substituted with one or more R sub groups. R3 and R5 are each independently CN or C1-C6 alkylene-CN, C(O)OR 13 or C1-C6 alkylene-C(O)OR 13 , S(O)OR 13 or C1-C6 alkylene-S(O)OR 13 , C(O)N(R’)(R”) or C1-C6 alkylene-C(O)N(R’)(R”), each of which may optionally be substituted with one or more R sub groups. R2 and R3, together with the adjacent carbon atoms, may form another ring of 3 to 7 atoms. R4 and R5, together with the adjacent carbon atoms, may form another ring of 3 to 7 atoms. R6 and R7, together with the adjacent carbon atoms, may form another ring of 3 to 7 atoms. R8 and R9, together with the adjacent carbon atoms, may form another ring of 3 to 7 atoms. R2, R3, R4, R5, R6, R7, R8, R9, R 14 and R 15 Two or more of may, together with the adjacent carbon atoms, form one or more rings of 5 to 7 atoms condensed with a ring containing two sulfur atoms.

[0221] [Linking coupling group] is: [Chemical formula] may have the structure of.

[0222] In formulas (P-I) and (P-II): [Chemical formula] represents a bond to the [cyclic disulfide moiety]; X1 and Z1 are each independently H, OH, OM, OR 13 , SH, SM, SR 13 , C(O)H, S(O)H or alkyl (each of which may optionally be substituted with one or more R sub groups), N(R’)(R”), B(R 13 )3, BH3 - , Se; or D-Q, where D is independently absent, O, S, N(R’), alkylene in each case, each of which may optionally be substituted with one or more R sub groups, and Q is independently a nucleoside or oligonucleotide in each case; X2 and Z2 are each independently N(R’)(R”), OR 18or D-Q, where D is independently in each case absent, O, S, N, N(R'), alkylene, each of which may optionally be substituted with one or more R sub groups, and Q is independently in each case a nucleoside or oligonucleotide, Y1 is S, O or N(R'); M is an organic or inorganic cation; and R 18 is H or alkyl optionally substituted with one or more R sub groups. X1 and Z1 may each independently be NSO2R' or N=CN(R')(R").

[0223] In certain embodiments, [linker coupling group] is

Chemical formula

[0224] In certain embodiments, [linker coupling group] has one of the following structures:

Chemical formula

[0225] In certain embodiments, [linker coupling group] is

Chemical formula

[0226] In certain embodiments, [linker coupling group] is

Chemical formula

[0227] In certain embodiments, [linker coupling group] is

Chemical formula

[0228] In certain embodiments, [linker coupling group] has the structure -P(Z)(X), where: X is selected from the group consisting of -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH2CH(CH3)2, -OCH2CH2CN, -OCH2CH2Si(CH3)3, -OCH2CH2Si(CH2CH3)3, -OC(H)=CH2, -OCH2C(H)=CH2

Chemical formula

Chemical formula

Chemical formula

[0229] In certain embodiments, [linker coupling group] is

Chemical formula

[0230] In certain embodiments, [linker coupling group] has various modifications for stabilization and has one of the following structures:

Chemical formula

Chemical formula

Chemical formula

[0231] Examples of the above compounds with different stabilities by phosphorus-containing nucleotide internucleotide linkages are as follows:

Table 23

[0232] [Cyclic disulfide moiety] has the structure

Chemical formula

[0233] Examples of [cyclic disulfide moiety] for the 5-membered cyclic compound of formula (C-I) are:

Chemical formula

[0234] In one embodiment, the compound is [Chemical formula] of the formula: where R2, R3, R4, and R5 are each independently H, alkyl (e.g., CH3), heterocyclic, CH2R 15 , aryl (e.g., phenyl), heteroaryl, CHFR 15 , CF2R 15 , CF3; and may be in any stereoisomeric configuration; and R 15 is alkyl, heterocyclic, aryl, OH, O-alkyl, NH2, NH(alkyl), N(alkyl)2, CF2R 15 or CF3; and may be in any stereoisomeric configuration.

[0235] In one embodiment, the compound is [Chemical formula] of the formula: where R2 and R3 are each independently H, alkyl (e.g., CH3), heterocyclic, CN, CF3, CH2R 15 , heteroaryl, CHFR 15 , CF2R 15 , C(O)NHR 15 , C(O)N(R 15 )2, C(O)OR 15 , S(O)OR 15 , CH2C(O)OR 15 , CH2S(O)OR 15 ; and may be in any stereoisomeric configuration; and R 15 is H, alkyl, heterocyclic, aryl (e.g., substituted or unsubstituted phenyl), heteroaryl, or CF3; and may be in any stereoisomeric configuration.

[0236] In one embodiment, the compound is: [Chemical formula] having the formula, where n is 1, 2, 3 or 4; R2 and R3 are each independently H, alkyl (e.g., CH3), heterocyclic, CN, CF3, CH2R 15 , aryl (e.g., substituted or unsubstituted phenyl), heteroaryl, CHFR 15 , CF2R 15 , C(O)NHR 15 , C(O)N(R 15 )2, C(O)OR 15 , S(O)OR 15 , CH2C(O)OR 15 , CH2S(O)OR 15 ; and may be in any stereoconfiguration; and R 15 is H, alkyl, heterocyclic, aryl (e.g., phenyl), heteroaryl or CF3; and may be in any stereoconfiguration.

[0237] Examples of compounds of formula (I) having a 5-membered cyclic disulfide moiety are shown in Table 1.

Table 24

Table 25

Table 26

Table 27

Table 28

[0238] In certain embodiments, [the cyclic disulfide moiety] has the structure

Chemical formula

[0239] In one embodiment, the compound is: [Chemical formula] and has, where n is 0, 1 or 2; m is 1, 2 or 3; R a , R b , R c , R d , R e and R f are each independently H, alkyl (e.g., CH3), heterocyclic, CF3, CH2R 15 , aryl (e.g., substituted or unsubstituted phenyl), heteroaryl, CHFR 15 , CF2R 15 , C(O)NHR 15 , C(O)N(R 15 )2, C(O)OR 15 , S(O)OR 15 , CH2C(O)OR 15 , CH2S(O)OR 15 ; and can be in any stereoisomeric configuration; and R 15 is H, alkyl, heterocyclic, aryl (e.g., substituted or unsubstituted phenyl), heteroaryl, hydroxyl, alkoxy, NH2, NH alkyl, NH(alkyl)2 or CF3; and can be in any stereoisomeric configuration.

[0240] Examples of the [cyclic disulfide moiety] of the bicyclic compound of formula (C-I) are: [Chemical formula] including.

[0241] In one embodiment, the compound is [Chemical formula] and has the formula, where R3 and R5 together with the adjacent carbon atoms and two sulfur atoms form a second ring (e.g., having 6 - 8 atoms).

[0242] Examples of the compounds of formula (I) having a bicyclic disulfide moiety are shown in Table 2.

Table 29

[0243] [Cyclic disulfide moiety] has the structure

Chemical formula

Chemical formula

Chemical formula

[0244] In certain embodiments, the compound is

Chemical formula

[0245] Examples of compounds of formula (I) having a large (7-membered or larger) cyclic disulfide moiety are shown in Table 3.

Table 30

[0246] [Cyclic disulfide moiety] has the structure [Chemical formula] may also have. Examples of [cyclic disulfide moiety] for the six-membered cyclic compound of formula (C-III) are: [Chemical formula] including.

[0247] In certain embodiments, the compound is [Chemical formula] has the formula of. In this formula, R2, R3, R4, R5 and R6 are each independently H, alkyl (e.g., CH3), heterocyclic, CH2R 15 , aryl (e.g., phenyl), heteroaryl, CHFR 15 , CF2R 15 , CF3; and may be in any stereoconfiguration; and R 15 is alkyl, heterocyclic, aryl, OH, O-alkyl, NH2, NH(alkyl), N(alkyl)2, CF2R 15 or CF3; and may be in any stereoconfiguration.

[0248] Examples of compounds of formula (I) having a six-membered cyclic disulfide moiety are shown in Table 4. [Table 31]

[0249] Throughout this specification, certain terms within chemical structures are abbreviated as is well known to those skilled in the art, including, for example, methyl (Me), benzoyl (Bz), phenyl (Ph) and pivaloyl (Piv).

[0250] The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine or iodine.

[0251] As used herein, the term "aliphatic" or "aliphatic group" means a straight-chain or branched, substituted or unsubstituted hydrocarbon chain that is saturated or contains one or more unsaturated units, or a monocyclic hydrocarbon or bicyclic or polycyclic hydrocarbon that is saturated or contains one or more unsaturated units but is not aromatic, having one point of attachment to the remainder of the molecule. In certain embodiments, the aliphatic group contains from 1 to 50 aliphatic carbon atoms, for example, from 1 to 10 aliphatic carbon atoms, from 1 to 6 aliphatic carbon atoms, from 1 to 5 aliphatic carbon atoms, from 1 to 4 aliphatic carbon atoms, from 1 to 3 aliphatic carbon atoms or from 1 to 2 aliphatic carbon atoms. In certain embodiments, "cycloaliphatic" refers to a monocyclic or bicyclic C3-C 10 hydrocarbon (e.g., a monocyclic C3-C6 hydrocarbon) that is saturated or contains one or more unsaturated units but is not aromatic and has one point of attachment to the remainder of the molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and their hybrids such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0252] The term "alkyl" refers to a hydrocarbon chain that can be straight-chain or branched-chain and contains the indicated number of carbon atoms. For example, C1-C 12 alkyl indicates that the group can have from 1 to 12 (including both ends) carbon atoms within the group. Unless otherwise specified, "alkyl" generally refers to C1-C 24 alkyl (e.g., C1-C 12refers to alkyl, C1-C8 alkyl or C1-C4 alkyl). The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes an alkyl moiety in which all hydrogens are replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may optionally contain 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.

[0253] 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). Examples of typical alkenyl 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 having 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 include ethynyl, 2-propynyl and 3-methylbutynyl and propargyl. sp 2 and sp 3 Carbon can, if desired, serve as the point of attachment for the alkenyl and alkynyl groups, respectively.

[0254] The term "alkoxy" refers to an -O-alkyl radical. The term "alkylene" refers to a divalent alkyl (i.e., -R-). The term "aminoalkyl" refers to an alkyl substituted with amino. The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical.

[0255] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" refers to a polymethylene group, i.e., -(CH2) n -(where n is a positive integer, preferably 1-6, 1-4, 1-3, 1-2 or 2-3). A substituted alkylene chain refers to a polymethylene group in which one or more methylene hydrogen atoms are replaced by substituents. Suitable substituents include those described below.

[0256] 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 by substituents. Suitable substituents include those described below.

[0257] 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 substituents. The term "aryl" may be used interchangeably with the term "aryl ring". Examples of aryl groups include phenyl, biphenyl, naphthyl, anthracyl, etc., which may have one or more substituents. Also included in 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 the term "aralkyl" refers to an alkyl substituted by aryl. The term "arylalkoxy" refers to an alkoxy substituted by aryl.

[0258] The term "cycloalkyl" or "cyclic" as used herein refers to a cyclic hydrocarbon group having 3-12 carbons, such as 3-8 carbons, such as 3-6 carbons, which is saturated and partially unsaturated but not aromatic, where the cycloalkyl group may optionally be further substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl.

[0259] The term "heteroaryl" or "heteroal-" refers 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, said heteroatoms being selected from O, N or S (e.g., if monocyclic, bicyclic or tricyclic respectively, carbon atoms and 1 to 3, 1 to 6 or 1 to 9 N, O or S heteroatoms), where 0, 1, 2, 3 or 4 atoms of each ring may be substituted by substituents. The term also includes groups in which the heteroaromatic ring is fused to one or more aryl, cycloalkyl or heterocyclyl rings and the linking radical or point is the heteroaromatic ring. Examples of heteroaryl groups include 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, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido[2,3-b]-1,4-oxazin-3(4H)-one and the like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted by heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted by heteroaryl.

[0260] The terms "heterocyclyl", "heterocycle", "heterocyclic radical", or "heterocyclic ring" 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., if monocyclic, bicyclic, or tricyclic, respectively, carbon atoms and 1 to 3, 1 to 6, or 1 to 9 heteroatoms of N, O, or S), where 0, 1, 2, or 3 atoms of each ring may be substituted with substituents. When used with reference to the ring atoms of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (such as in 3,4-dihydro-2H-pyrrolyl), NH (such as in pyrrolidinyl), or +NR (such as in N-substituted pyrrolidinyl). Examples of heterocyclyl groups include triazolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, where the alkyl and heterocyclyl moieties are independently optionally substituted as desired.

[0261] The term "oxo" refers to an oxygen atom that forms a carbonyl when bonded to carbon, an N-oxide when bonded to nitrogen, and a sulfoxide or sulfone when bonded to sulfur.

[0262] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted with substituents.

[0263] The term "substituted" refers to the replacement of one or more hydrogen radicals in a structure with radicals of specific substituents, 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 substituents may be further substituted.

[0264] Suitable divalent substituents for the saturated carbon atoms of a "optionally substituted" group include the following: =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 in each case hydrogen, C 1-6 aliphatic which may be substituted as defined below or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur). Suitable divalent substituents bonded to the vicinal substitutable carbon of an "optionally substituted" group are -O(CR * 2) 2-3 O- (where R *is independently, in each case, hydrogen, a C1-6 aliphatic which may be substituted as defined below or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur).

[0265] Stereoisomers and chiral purity of enriched compounds Certain compounds of the invention may exist, in particular, in geometric or stereoisomeric forms. The invention is intended to embrace all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof and other mixtures thereof, falling within the scope of the invention. Further asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are intended to be included within the present invention.

[0266] For example, when a particular enantiomer of a compound is desired, it can be prepared by asymmetric synthesis or by separation of the resulting mixture of diastereomers and derivatization from a chiral auxiliary by cleavage of the auxiliary group to afford the desired enantiomer in chiral pure / enriched form. Alternatively, when the molecule contains a basic functional group such as amino or an acidic functional group such as carboxyl, a diastereomeric salt is formed with a suitable optically active acid or base and the thus formed diastereomers are subsequently resolved by fractional crystallization or chromatography means well known in the art and the chiral pure / enriched enantiomer is then recovered.

[0267] Certain embodiments of the invention include oligonucleotides that are substantially chiral pure or chiral enriched at specific positions within the oligonucleotide. Examples of substantially chiral pure oligonucleotides include those having phosphorothioate linkages that are at least 75% Sp or Rp (Cook et al., U.S. Patent 5,587,361) and those having substantially chiral pure (Sp or Rp) alkylphosphonate, phosphoramidate or phosphotriester linkages (Cook, U.S. Patents 5,212,295 and 5,521,302), but are not limited thereto.

[0268] The chiral purity with respect to the chiral linking phosphorus atom for each terminal, chirally modified nucleotide - nucleotide linkage is at least 50%, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or virtually 100%.

[0269] A chiral - pure (or substantially chiral - pure) diastereoisomeric form of a compound or oligonucleotide refers to a specific diastereoisomeric form of a compound or oligonucleotide having a chiral purity of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or virtually 100%.

[0270] Accordingly, embodiments of the present invention provide structures of formula (I) or (II), formula (C - I), (C - IIa) or (C - IIb), formula (P - I) or (P - II) that are present in a chiral - pure or enriched diastereoisomeric form having a chiral purity of at least at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or virtually 100%.

[0271] Examples of chiral - pure / enriched compounds are as follows: [Table 32] [Table 33]

[0272] Oligonucleotide prodrugs Another aspect of the present invention relates to an oligonucleotide (e.g., a single-stranded iRNA agent or a double-stranded iRNA agent) comprising one or more compounds comprising the structure of formula (I): [Cyclic disulfide moiety]-[Linker coupling group] (I). In formula (I), at least one [Linker coupling group] comprises a nucleoside or an oligonucleotide.

[0273] All of the formulas of the [Cyclic disulfide moiety], all of the formulas of the [Linker coupling group], all of the variable groups defined by these formulas, and all of the subgeneric and species structures related to this compound, all of the above embodiments related to the [Cyclic disulfide moiety] and [Linker coupling group] (or modified phosphate prodrug compound) in the first aspect of the present invention related to this compound are applicable to this aspect of the present invention related to oligonucleotides.

[0274] In certain embodiments, the oligonucleotide comprises at least one [Cyclic disulfide moiety] at the 5'-end of the oligonucleotide.

[0275] In certain embodiments, the oligonucleotide comprises at least one [Cyclic disulfide moiety] at the 3'-end of the oligonucleotide.

[0276] In certain embodiments, the oligonucleotide comprises at least one [Cyclic disulfide moiety] at an internal position of the oligonucleotide.

[0277] In certain embodiments, when the [Cyclic disulfide moiety] has the structure of formula (C-III), at least one [Cyclic disulfide moiety] is attached to the 5'-end of a nucleoside or an oligonucleotide.

[0278] Further structures of the modified phosphate prodrug compounds include those disclosed in WO2014 / 088920 published on June 12, 2014, the content of which is incorporated herein by reference in its entirety. In particular, these modified phosphate prodrug compounds are incorporated into the oligonucleotide at the 5'-end.

[0279] In certain embodiments, the oligonucleotide is a single-stranded oligonucleotide such as a single-stranded iRNA agent (e.g., single-stranded siRNA).

[0280] In certain embodiments, the oligonucleotide is a double-stranded oligonucleotide such as a double-stranded iRNA agent (e.g., double-stranded siRNA) that includes a sense strand and an antisense strand.

[0281] In certain embodiments, the sense strand includes at least one [cyclic disulfide moiety]. In certain embodiments, the antisense strand includes at least one [cyclic disulfide moiety]. In certain embodiments, both the sense strand and the antisense strand each include at least one [cyclic disulfide moiety].

[0282] The introduction of the [cyclic disulfide moiety] to the phosphate group as a temporary protecting group to the sense strand or the antisense strand or both the sense strand and the antisense strand is shown in Schemes 10-15 of Example 9 below.

[0283] Oligonucleotide definition and design Unless otherwise defined, the nomenclature, methods, and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceuticals and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Some such techniques and methods are described, for example, in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 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 hereby incorporated by reference for all purposes. If permitted, all patents, applications, published applications, and other compounds and other data cited throughout this disclosure are hereby incorporated by reference in their entirety.

[0284] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be regulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding a target protein (including, but not limited to, pre-mRNA and mRNA or portions thereof) and also cDNA derived from such RNA and miRNA. For example, a target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state. In certain embodiments, the target nucleic acid can be a nucleic acid molecule from an infectious agent.

[0285] As used herein, the term "iRNA" refers to an agent that mediates cleavage of a target RNA transcript. These agents bind to a cytoplasmic multiprotein complex known as the RNA interference-induced silencing complex (RISC). Agents that are effective in inducing RNA interference are also referred to herein as siRNA, RNAi agents or iRNA agents. Thus, these terms can be used interchangeably herein. The term iRNA as used herein includes microRNA and pre-microRNA. Furthermore, the "compound" of the invention as used herein refers to an iRNA agent and can be used interchangeably with an iRNA agent.

[0286] The iRNA agent should contain a region of sufficient homology to the target gene and be of sufficient length in terms of nucleotides so that the iRNA agent or its fragment can mediate down-regulation of the target gene. (For the sake of simplicity in explanation, the term nucleotide or ribonucleotide may sometimes be used herein to refer to one or more monomeric subunits of the iRNA agent. It is understood that the use of the term "ribonucleotide" or "nucleotide" herein may also refer to a modified nucleotide or a surrogate exchange moiety at one or more positions in the case of a modified RNA or a nucleotide surrogate.) Thus, the iRNA agent is or contains a region that is at least partially and in some embodiments completely complementary to the target RNA. There is no need for complete complementarity between the iRNA agent and the target, but the match must be sufficient for the iRNA agent or its cleavage product to be able to direct sequence-specific silencing, for example, by RNAi cleavage of the target RNA, such as mRNA. The degree of complementarity or homology to the target strand is most important in the antisense strand. In particular in the antisense strand, complete complementarity is often desired, but some embodiments may contain one or more or, for example, 6, 5, 4, 3, 2 or less mismatches (to the target RNA), especially in the antisense strand. The sense strand only needs to be sufficiently complementary to the antisense strand to maintain the overall double-stranded character of the molecule.

[0287] An iRNA agent is: a molecule long enough to induce an interferon response (cleaved by Dicer (Bernstein et al. 2001. Nature, 409: 363-366) and capable of entering RISC (RNAi-induced silencing complex)); and a molecule short enough not to induce an interferon response (this molecule is also cleaved by Dicer and / or can enter RISC), for example, a molecule of a size insertable into RISC, for example, a molecule mimicking a Dicer cleavage product. A molecule short enough not to induce an interferon response is herein referred to as an siRNA agent or a short iRNA agent. As used herein, "siRNA agent or short iRNA agent" refers to a sufficiently short iRNA agent that does not induce a harmful interferon response in human cells, for example, a double-stranded RNA agent or a single-stranded agent, for example, containing a double-stranded region of less than 60, 50, 40 or 30 nucleotide pairs. The siRNA agent or its cleavage product, for example, with respect to a target RNA, down-regulates a target gene by induction of RNAi, where the target includes endogenous or pathogen target RNA.

[0288] As used herein, a "single-stranded iRNA agent" is an iRNA agent consisting of a single molecule. It may contain a double-stranded region formed by intra-strand pairing, for example, may be or include a hairpin or panhandle structure. The single-stranded iRNA agent may be antisense to a target molecule. The single-stranded iRNA agent is of a length sufficient to enter RISC and participate in RISC-mediated cleavage of the target mRNA. The single-stranded iRNA agent is at least 14 nucleotides in length and in some 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.

[0289] A loop refers to a region of an iRNA strand that is unpaired with the opposite nucleotides in a double-strand when a section of the iRNA strand forms base pairs with another strand or another section of the same strand.

[0290] The hairpin iRNA agent has a double-stranded region of exactly or at least 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs. The double-stranded region can be 200, 100 or 50 in length or less. In certain embodiments, the double-stranded region ranges from 15 to 30, 17 to 23, 19 to 23 and 19 to 21 nucleotide pairs in length. The hairpin can have a single-stranded overhang or terminal unpaired region, in certain embodiments at the 3' and in certain embodiments on the antisense side of the hairpin. In certain embodiments, the overhang is 2 to 3 nucleotides in length.

[0291] As used herein, "double-stranded (ds) iRNA agent" refers to an iRNA agent comprising more than one and in some cases two strands, where intermolecular hybridization can form a region of double-stranded structure.

[0292] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0293] As used herein, "gene silencing" by an RNA interference molecule refers to a reduction in the mRNA level for a target gene in a cell of 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% to a maximum of 100% (including 100%) and any integer therebetween relative to the mRNA level seen in the cell in the absence of the miRNA or RNA interference molecule. In preferred embodiments, 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% to a maximum of 100% (including 100%) and any integer between 5% and 100%.

[0294] As used herein, the term "regulating gene expression" means that the level of gene expression or of an RNA molecule encoding one or more proteins or protein subunits or an equivalent RNA molecule is upregulated or downregulated such that it exceeds or is less than that observed in the absence of a modulator. For example, the term "regulating" may include "inhibiting", but the use of the term "regulating" is not limited to this definition.

[0295] Gene expression regulation as used herein occurs when the level of gene expression or of an RNA molecule encoding one or more proteins or protein subunits or an equivalent RNA molecule is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more different 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, e.g., as follows.

Number

[0296] In connection with gene expression, as used herein, the terms "inhibiting", "downregulating" or "decreasing" mean that the level of gene expression or of an RNA molecule encoding one or more proteins or protein subunits or an equivalent RNA molecule or the activity of one or more proteins or protein subunits is decreased below that observed in the absence of a modulator. Gene expression is downregulated when the level of gene expression or of an RNA molecule encoding one or more proteins or protein subunits or an equivalent RNA molecule or the activity of one or more proteins or protein subunits is decreased by at least 10%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or most preferably 100% (i.e., no gene expression) relative to a corresponding unregulated control.

[0297] In the context of gene expression, the terms "increased" or "upregulated" as used herein mean that the level of gene expression or an RNA molecule encoding one or more proteins or protein subunits or an equivalent RNA molecule or the activity of one or more proteins or protein subunits is increased beyond what is observed in the absence of a modulator. Gene expression is upregulated when the level of gene expression or an RNA molecule encoding one or more proteins or protein subunits or an equivalent RNA molecule or the activity of one or more proteins or protein subunits is increased by at least 10%, 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 more as compared to the corresponding unregulated control.

[0298] The terms "increased" or "increase" as used herein generally mean an increase in a statistically significant amount; to avoid any doubt, "increase" means an increase of at least 10% compared to a reference level, for example an increase of 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 a maximum of 100% (including 100%) or any increase from 10 - 100% compared to the reference level or an increase of at least about 2-fold or at least about 3-fold or at least about 4-fold or at least about 5-fold or at least about 10-fold or an increase of 2-fold - 10-fold or more compared to the reference level.

[0299] As used herein, the terms "reduced" or "reduction" generally mean a statistically significant amount of reduction. However, to avoid doubt, "reduction" means a reduction of at least 10% compared to the 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 100% (including 100%) reduction (i.e., absence level compared to the reference sample) or any reduction from 10 to 100% compared to the reference level.

[0300] Double-stranded iRNA comprises two oligonucleotide strands that are sufficiently complementary to hybridize and form a double-stranded structure. Generally, the double-stranded structure is 15 to 30, more generally 18 to 25, even more generally 19 to 24 and most generally 19 to 21 base pairs in length. In certain embodiments, a long double-stranded iRNA of 25 to 30 base pairs in length is preferred. In certain embodiments, a short double-stranded iRNA of 10 to 15 base pairs in length is preferred. In other embodiments, the double-stranded iRNA is at least 21 nucleotides in length.

[0301] In certain embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, where the antisense RNA strand has a region of complementarity that is complementary to at least a portion of the target sequence and the double-stranded region is 14 to 30 nucleotides in length. Similarly, the region of complementarity to the target sequence is 14 to 30, more generally 18 to 25, even more generally 19 to 24 and most generally 19 to 21 nucleotides in length.

[0302] As used herein, the term "antisense strand" 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 and 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.

[0303] The term "sense strand" refers to an oligonucleotide strand that has the same nucleoside sequence as a target sequence, such as the sequence of messenger RNA or DNA, either in whole or in part. The terms "sense strand" and "messenger strand" are used interchangeably herein.

[0304] "Specifically hybridizable" and "complementary" mean that a nucleic acid can hydrogen bond with another nucleic acid sequence in a traditional Watson-Crick or other non-traditional type. For the nucleic acid molecules of the present invention, the binding free energy of the nucleic acid molecule and its complementary sequence is sufficient to enable the nucleic acid to perform related functions, such as RNAi activity. The determination of the binding free energy of nucleic acid molecules 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, / . Am. Chem. Soc. 109: 3783-3785). Percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5 out of 10, 6, 7, 8, 9, 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "Fully complementary" or 100% complementarity means that all contiguous residues of a nucleic acid sequence hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than complete complementarity refers to a situation where some, but not all, of the nucleoside units of the two strands can hydrogen bond to each other. "Substantial complementarity" refers to a polynucleotide strand that exhibits 90% or more complementarity, excluding regions in the polynucleotide strand such as overhangs that are selected to be non-complementary. Specific binding requires a degree of complementarity sufficient to avoid non-specific binding of the oligonucleotide to non-target sequences under the conditions where specific binding is desired, i.e., under physiological conditions in an in vivo assay or a therapeutic treatment or under the conditions under which the assay is performed in an in vitro assay. Non-target sequences typically differ by at least 5 nucleotides.

[0305] In certain embodiments, the double-stranded region is exactly or at least 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.

[0306] In certain embodiments, the antisense strand is exactly or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0307] In certain embodiments, the sense strand is exactly or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0308] In certain embodiments, the sense and antisense strands are each 15-30 nucleotides in length. In certain embodiments, the sense and antisense strands are each 19-25 nucleotides in length. In certain embodiments, the sense and antisense strands are each 21-23 nucleotides in length.

[0309] In certain embodiments, one strand has at least one stretch of 1-5 single-stranded nucleotides in the double-stranded region. A "stretch of single-stranded nucleotides in the double-stranded region" means that there are at least one nucleotide base pair at both ends of the single-stranded stretch. In certain embodiments, both strands have at least one stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. When both strands have a stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region, such single-stranded nucleotides can be in opposite directions (e.g., a stretch of mismatches) or the second strand can be positioned such that it does not have single-stranded nucleotides opposite to those of the single-stranded iRNA of the first strand, and vice versa (e.g., a single-stranded loop). In certain embodiments, the single-stranded nucleotides are within 8 nucleotides from either end, from the 5' or 3' end of the complementary region between the two strands, e.g., 8, 7, 6, 5, 4, 3, or 2 nucleotides.

[0310] In certain embodiments, the oligonucleotide comprises a single-stranded overhang at at least one end. In certain embodiments, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0311] In certain embodiments, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein these strands form a double-stranded region of 21 contiguous base pairs having a 2-nucleotide-long single-stranded overhang at the 3' end.

[0312] 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.

[0313] In certain embodiments, the two strands of the double-stranded oligonucleotide can bind to each other. The two strands can bind to each other at both ends or only at one end. Binding at one end refers to the 5' end of the first strand binding to the 3' end of the second strand or the 3' end of the first strand binding to the 5' end of the second strand. When the two strands bind to each other at both ends, the 5' end of the first strand binds to the 3' end of the second strand and the 3' end of the first strand binds to the 5' end of the second strand. The two strands can be bound to each other by an oligonucleotide linker comprising, but not limited to, (N) n (wherein N is independently a modified or unmodified nucleotide and n is from 3 to 23). In certain embodiments, n is from 3 to 10, for example, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can participate in base pair interactions with other nucleotides of the linker. The two strands can also be bound to each other by a non-nucleoside linker, such as the linkers described herein. One of ordinary skill in the art will recognize that any of the oligonucleotide chemical modifications or variations described herein can be used for the oligonucleotide linker.

[0314] Hairpin and dumbbell-shaped oligonucleotides have duplex regions of exactly or at least 14, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs. The duplex region can be 200, 100 or 50 bases 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.

[0315] Hairpin oligonucleotides can have single-stranded overhangs or terminal unpaired regions in certain embodiments at the 3' end and in certain embodiments on the antisense side of the hairpin. In certain embodiments, the overhang is 1-4, more generally 2-3 nucleotides in length. Hairpin oligonucleotides capable of inducing RNA interference are also referred to herein as "shRNA".

[0316] In certain embodiments, two oligonucleotide strands specifically hybridize if they have a degree of complementarity sufficient to avoid non-specific binding of the antisense strand to non-target nucleic acid sequences under the conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments or under the conditions under which the assay is performed in the case of in vitro assays.

[0317] As used herein, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which the antisense strand hybridizes to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. The "stringent conditions" under which the antisense strand hybridizes to the target sequence are determined by the nature and composition of the antisense strand and the assay being tested.

[0318] It is understood by those skilled in the art that the incorporation of nucleotide affinity modifications tolerates a greater number of mismatches compared to unmodified oligonucleotides. Similarly, certain oligonucleotide sequences are 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, such as by determination of the melting temperature (Tm). Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, by the techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443), those skilled in the art can evaluate nucleotide modifications with respect to their ability to raise the melting temperature of an RNA:DNA duplex.

[0319] Further dsRNA design In certain embodiments, the oligonucleotide is an iRNA agent, the iRNA agent is a 19 nt long double ended bluntmer, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5' end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0320] In certain embodiments, the oligonucleotide is an iRNA agent, the iRNA agent is a 20 nt long double ended bluntmer, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5' end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0321] In certain embodiments, the oligonucleotide is an iRNA agent, the iRNA agent is a 21 nt long blunt-ended duplex, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10 and 11 counting from the 5'-end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12 and 13 counting from the 5'-end.

[0322] In certain embodiments, the oligonucleotide is an iRNA agent, 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 counting from the 5'-end; the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12 and 13 counting from the 5'-end, wherein one end of the iRNA is blunt and the other end comprises a 2 nt overhang. Preferably, the 2 nt overhang is at the 3'-end of the antisense. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0323] In one embodiment, the oligonucleotide is an iRNA agent, the iRNA agent comprising a sense strand and an antisense strand, wherein: the sense strand is 25-30 nucleotide residues in length, wherein positions 1-23 of the first strand starting from the 5'-terminal nucleotide (position 1) comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and comprises at least 8 ribonucleotides at a position that pairs with positions 1-23 of the sense strand starting from the 3'-terminal nucleotide, forming a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and a maximum of 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3'-single-stranded overhang of 1-6 nucleotides; wherein the 5'-terminal of the antisense strand comprises 10-30 consecutive nucleotides that are unpaired with the sense strand, thereby forming a single-stranded 5'-overhang of 10-30 nucleotides; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand base pair with nucleotides when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; and the antisense strand is targeted to be fully complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression when the double-stranded nucleic acid is introduced into mammalian cells; wherein the sense strand comprises at least one motif that is three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near a cleavage site. The antisense strand comprises at least one motif that is three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site

[0324] In certain embodiments, the oligonucleotide is an iRNA agent, the iRNA agent comprises a sense strand and an antisense strand, wherein the iRNA agent has a first strand of at least 25 and a maximum of 29 in length and a second strand having a length of a maximum of 30 nucleotides, and has at least one motif which is three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 at the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer at the 3' end than the first strand, and wherein the double-stranded region which is at least 25 nucleotides in length and the second strand are sufficiently complementary to the target mRNA along at least 19 nt of the second strand length to reduce target gene expression when the iRNA agent is introduced into mammalian cells, and wherein the dicer cleavage of the iRNA preferentially yields siRNA comprising the 3' end of the second strand, whereby mammalian target gene expression is reduced. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0325] In certain embodiments, the sense strand comprises at least one motif which is three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at the cleavage site in the sense strand. For example, the sense strand may comprise at least one motif which is three 2'-F modifications on three consecutive nucleotides within positions 7 to 15 from the 5' end.

[0326] In certain embodiments, the antisense strand may also comprise at least one motif which is three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at or near the cleavage site of the antisense strand. For example, the antisense strand may comprise at least one motif which is three 2'-O-methyl modifications on three consecutive nucleotides within positions 9 to 15 from the 5' end.

[0327] For iRNA agents having a double-stranded region 17 - 23 nt in length, the cleavage sites of the antisense strand are typically approximately at positions 10, 11, and 12 from the 5' end. Thus, three identical modified motifs occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleotide from the 5' end of the antisense strand or the count starting from the first paired nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the iRNA from the 5' end.

[0328] In certain embodiments, the oligonucleotide is an iRNA agent, and the iRNA agent comprises a sense strand and an antisense strand each having 14 - 30 nucleotides, where the sense strand comprises at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand, and at least one of the motifs occurs in another part of the strand separated from the motif by at least one nucleotide at the cleavage site. In certain embodiments, the antisense strand also comprises at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand. The modification in the motif occurring at or near the cleavage site of the sense strand is different from the modification in the motif occurring at or near the cleavage site of the antisense strand.

[0329] In certain embodiments, the oligonucleotide is an iRNA agent, and the iRNA agent comprises a sense strand and an antisense strand each having 14 - 30 nucleotides, where the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site of the strand. In certain embodiments, the antisense strand also comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0330] In certain embodiments, the oligonucleotide is an iRNA agent, and the iRNA agent comprises a sense strand and an antisense strand each having 14 to 30 nucleotides, 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 wherein 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.

[0331] In certain embodiments, the oligonucleotide is an iRNA agent, and the iRNA agent comprises mismatches with the target within the duplex or combinations thereof. Mismatches can occur in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of binding or dissociation of a particular pairing; the simplest approach is to test the pairs on an individual pair basis, but next-nearest neighbor or similar analyses can also be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings containing universal bases are preferred over canonical pairings.

[0332] In certain embodiments, the oligonucleotide is an iRNA agent, and the iRNA agent independently comprises at least one selected from A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, in the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which promotes dissociation of the antisense strand at the 5' end of the duplex.

[0333] In certain embodiments, the nucleotide at position 1 within 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 within the double-stranded region from the 5'-end of the antisense strand is an AU base pair. For example, the first base pair within the double-stranded region from the 5'-end of the antisense strand is an AU base pair.

[0334] In certain 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 50% of the dsRNA agent is modified, 50% of all the nucleotides present in the dsRNA agent contain the modifications described herein.

[0335] 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'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F.

[0336] In certain embodiments, each of the sense strand and the antisense strand is independently modified with non-natural nucleotides such as acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0337] In certain embodiments, each of the sense strand and the antisense strand of the dsRNA agent comprises at least two different modifications.

[0338] In certain 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.

[0339] In certain embodiments, the oligonucleotide does not contain any 2'-F modifications.

[0340] 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 of the sense strand or the antisense strand or both, at any position of the strand. For example, the internucleotide linkage modification may occur at all nucleotides of 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 internucleotide linkage modifications in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of internucleotide linkage modifications in the sense strand may be shifted relative to the alternating pattern of internucleotide linkage modifications in the antisense strand.

[0341] In certain embodiments, the oligonucleotide is an iRNA agent, and the iRNA comprises phosphorothioate or methylphosphonate internucleotide linkage modifications in the overhang region. For example, the overhang region can include two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. The internucleotide linkage modification can also be formed to bind the overhang nucleotide to the terminal paired nucleotide within the duplex region. For example, at least two, three, four, or all of the overhang nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkages, and optionally, there can be additional phosphorothioate or methylphosphonate internucleotide linkages that bind the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third pairs with the nucleotide adjacent to the overhang nucleotide. Preferably, these terminal three nucleotides can be the 3' end of the antisense strand.

[0342] In certain embodiments, the sense strand and / or the antisense strand comprises 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.

[0343] In certain embodiments, each of the sense strand and the antisense strand has 15 to 30 nucleotides. In one example, the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 25 nucleotides. In other examples, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0344] In certain embodiments, the nucleotide at the 1st position at the 5' end of the antisense strand of the duplex is selected from the group consisting of A, dA, dU, U, and dT. In certain embodiments, at least one of the first, second, and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0345] In certain embodiments, the antisense strand of the dsRNA agent is 100% complementary to the target RNA to be hybridized and inhibits its expression via 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.

[0346] In one aspect, the present invention relates to oligonucleotides (e.g., dsRNA agents) as defined herein that can inhibit the expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each having 14 to 40 nucleotides. The sense strand includes at least one destabilizing nucleotide, wherein at least one of the destabilizing 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).

[0347] The destabilizing nucleotide can occur, for example, at positions 14 to 17 at the 5' end of the sense strand when the sense strand is 21 nucleotides in length. The antisense strand contains at least two modified nucleic acids smaller than the sterically demanding 2'-OMe modification. Preferably, the two modified nucleic acids smaller than the sterically demanding 2'-OMe are 11 nucleotides apart. For example, the two modified nucleic acids are at positions 2 and 14 at the 5' end of the antisense strand.

[0348] In certain embodiments, the oligonucleotide is a dsRNA agent, and the dsRNA agent is: (a) (i) 18 to 23 nucleotides in length; (ii) having three consecutive 2'-F modifications at positions 7 to 15 of the sense strand; and (b) (i) 18 to 23 nucleotides in length; (ii) having at least one 2'-F modification somewhere in the strand; and (iii) having at least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides of the antisense strand wherein the dsRNA agent has one or more lipophilic monomers conjugated to one or more locations on at least one strand and containing one or more lipophilic moieties; and has a 2 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 double strand.

[0349] In certain embodiments, the oligonucleotide is a dsRNA agent, and the dsRNA agent is: (a) (i) 18 to 23 nucleotides in length; (ii) having less than 4 2'-F modifications of the sense strand; (b) (i) 18 to 23 nucleotides in length; (ii) having 12 or fewer 2'-F modifications; and (iii) At least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides in an antisense strand and the dsRNA agent has one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to one or more positions on at least one strand; and has a 2-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 double strand.

[0350] In certain embodiments, the oligonucleotide is a dsRNA agent, and the dsRNA agent: (a) (i) Is 19 to 35 nucleotides in length; (ii) Has less than 4 2'-F modifications in a sense strand; (b) (i) Is 19 to 35 nucleotides in length; (ii) Has 12 or fewer 2'-F modifications; and (iii) At least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides in an antisense strand and the double-stranded region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); where the dsRNA agent has one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to one or more positions on at least one strand; and has a 2-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 double strand.

[0351] In certain embodiments, the oligonucleotide is a dsRNA agent, the dsRNA agent comprising a sense strand and an antisense strand having a length of 15 to 30 nucleotides; the antisense strand comprising at least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides; wherein the double-stranded region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to one or more positions of at least one strand; and wherein the dsRNA agent has less than 20%, less than 15%, and less than 10% non-natural nucleotides.

[0352] In certain embodiments, the oligonucleotide is a dsRNA agent, the dsRNA agent comprising a sense strand and an antisense strand having a length of 15 to 30 nucleotides; the antisense strand comprising at least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides; wherein the double-stranded region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to one or more positions of at least one strand; and wherein the dsRNA agent has more than 80%, more than 85%, and more than 90% natural nucleotides, where 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides, for example.

[0353] In certain embodiments, the oligonucleotide is a dsRNA agent, the dsRNA agent comprising a sense strand and an antisense strand having a length of 15 to 30 nucleotides; the antisense strand comprising at least two phosphorothioate internucleotide linkages (counting from the 5' end) in the first 5 nucleotides; wherein the double-stranded region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to one or more positions of at least one strand; and wherein the dsRNA agent has 100% natural nucleotides, where 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides, for example.

[0354] In one embodiment, the oligonucleotide is a dsRNA agent, the dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand sequence is of formula (I): 5’ n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ (I) 〔wherein: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least 2 differently modified nucleotides; each N b represents an oligonucleotide sequence containing 1, 2, 3, 4, 5 or 6 modified nucleotides; each n p and n q each represent an overhang nucleotide; wherein N b and Y do not have the same modification; wherein XXX, YYY and ZZZ each independently represent one motif which is three identical modifications on three consecutive nucleotides.〕 represented by, wherein the dsRNA agent has one or more lipophilic monomers comprising one or more lipophilic moieties conjugated to one or more locations on at least one strand; and wherein the antisense strand of the dsRNA comprises two blocks of phosphorothioate nucleotide linkages separated by 1, 2, or 3 phosphate nucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 phosphate nucleotide linkages.

[0355] A variety of publications disclose multimeric iRNAs that can be used in the iRNAs of the present invention. Such publications include WO2007 / 091269, US Patent 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are hereby incorporated by reference in their entirety.

[0356] In certain embodiments, the antisense strand is 100% complementary to the target RNA to which it hybridizes and inhibits its expression via 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.

[0357] Double-stranded motif examples In certain embodiments, the oligonucleotide is a dsRNA agent, and the sense strand of the dsRNA agent has one of the following modification patterns. [Table 34] Here, n is a 2'-O-methyl-nucleotide; s is a phosphorothioate nucleotide internucleoside linkage; Nf is a 2'-fluoro-modified nucleotide; (Lipo) is n, Nf, or optionally a lipophilic-modified nucleotide (e.g., (Nhd)-2'-O-hexadecyl-modified nucleotide); Each (inv) is an inverted nucleotide (e.g., an inverted deoxynucleotide such as an inverted deoxyribonucleotide); and (L1) and at least one of (L2) contains a lipophilic group (e.g., C 10 -C 30 alkyl or C 10 -C 30 alkenyl group, e.g., C 16 alkyl, C 16Alkenyl, C 18 Alkyl, C 18 Alkenyl, C 20 Alkyl, C 20 Alkenyl, C 22 Alkyl, C 22 Alkenyl, C 24 Alkyl or C 24 a ligand containing alkenyl), and the other of (L1) and (L2) is absent or hydrogen.

[0358] When (Lipo) is absent, in any of the previous S1 - S23 modification patterns, the 3' or 5' end of the chain can be conjugated to a ligand (e.g., the targeting ligands described herein). In certain embodiments, the ligand is conjugated at the 5' end of the chain. In other embodiments, the ligand is conjugated at the 5' end of the chain. Examples of targeting ligands are described herein, for example, carbohydrate - based ligands that target liver tissue. In certain embodiments, the carbohydrate - based ligand is selected from the group consisting of galactose, polyvalent galactose, N - acetyl - galactosamine (GalNAc), polyvalent GalNAc, mannose, polyvalent mannose, lactose, polyvalent lactose, N - acetyl - glucosamine (GlcNAc), polyvalent GlcNAc, glucose, polyvalent glucose, fucose, and polyvalent fucose.

[0359] In certain embodiments, the oligonucleotide is a dsRNA agent, and the antisense strand of the dsRNA agent has one of the following modification patterns:

Table 35

[0360] In a further embodiment, Z has a [cyclic disulfide moiety] of the structure of formula (C-I) or (C-IIa) or (C-IIb) defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0361] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-Ia) defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0362] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-Ib) defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0363] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-Ic) defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0364] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-Id) defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0365] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-Ie) defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0366] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-If) as defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0367] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-IIa) as defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0368] In a further embodiment, Z comprises a [cyclic disulfide moiety] of the structure of formula (C-IIa) as defined above, attached to the 5'-end of the oligonucleotide via a [linker coupling group].

[0369] In a further embodiment, Z has a structure selected from the following and comprises a [cyclic disulfide moiety]:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0370] In each of the above embodiments, the [linker coupling group] is as defined in any of the above embodiments; for example, the [linker coupling group] can be attached at the 5'-carbon of the 5'-terminal nucleotide.

Chemical formula

[0371] In further embodiments of each of the above examples of the sense and antisense strands, each of the sense strands S1 - S23 can be duplexed with any of the antisense strands AS1 - AS17.

[0372] Nucleic acid modification In certain 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 modified internucleoside linkages, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modifications can be present anywhere in the oligonucleotide. For example, a modification can be present in one of the RNA molecules.

[0373] Nucleic acid modification (nucleobase) The naturally occurring base moieties of nucleosides are typically heterocyclic bases. Two of the most common classes of such heterocyclic bases are purines and pyrimidines. For nucleosides containing a pentofuranosyl sugar, a phosphate group can be attached to the 2', 3' or 5'-hydroxyl moiety of the sugar. In the formation of oligonucleotides, these phosphate groups are covalently bonded to adjacent nucleosides to form a linear polymeric compound. Within an oligonucleotide, the phosphate groups are generally said to form the internucleoside backbone of the oligonucleotide. The naturally occurring linkages or backbones of RNA and DNA are 3'→5' phosphodiester linkages.

[0374] 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 of skill in the art are applicable to the oligonucleotides described herein. Unmodified or natural nucleobases can be modified or replaced to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be made from any of these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nebularine, isoguanidine or tubercidin) and the oligomer modifications described herein. Alternatively, substitutions or modified analogs of any of the above bases and "universal bases" can be used. When natural bases are replaced with non-natural and / or universal bases, the nucleotides are said to have modified nucleobases and / or nucleobase modifications herein. Modified nucleobases and / or nucleobase modifications include natural, non-natural and universal bases, which include conjugate moieties, such as ligands described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups that can be conjugated to nucleobases via a linker having a suitable alkyl, alkenyl or amide bond.

[0375] The oligonucleotides described herein include nucleic acid base (often also simply referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleic acid bases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Examples of modified nucleic acid bases include other synthetic and natural nucleic acid bases such as inosine, xanthine, hypoxanthine, nebularine, isoguanidine, 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, 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-(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-diazol-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-(methoxycarbonyl-methyl)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, 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-(aminocarbonylethenyl)-pseudouracil, 1-(aminocarbonylethenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-2,4-(dithio)pseudouracil, 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaz)-2-(oxo)-phenothiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenothiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaz)-2-(oxo)-phenothiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxo)-naphthalene, inosine, xanthine, hypoxanthine, nubraline, tubercidin, isoguanidine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbolstyryl, 5-(methyl)isocarbolstyryl, 3-(methyl)-7-(propynyl)isocarbolstyryl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyridinyl, isocarbolstyryl, 7-(propynyl)isocarbolstyryl, propynyl-7-(aza)indolyl, 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 pyrimidine, N, 2 -substituted purine, N6 - Substitution purine, O 6 - Substitution purine, substitution 1,2,4-triazole, 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-alkylation or N-alkylation derivative thereof, including but not limited to. Alternatively, any substitution or modified analog of the above base and "universal base" can be used.

[0376] As used herein, a universal nucleobase refers to any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting melting behavior, recognition by intracellular enzymes, or the activity of the iRNA duplex. Some examples of universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbolstyryl, 5-methylisocarbolstyryl, 3-methyl-7-propynylisocarbolstyryl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyridinyl, isocarbolstyryl, 7-propynylisocarbolstyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylirinolyl, 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).

[0377] Additional nucleobases include those disclosed in U.S. Patent 3,687,808; those disclosed in International Application PCT / US09 / 038425 filed on March 26, 2009; those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858 - 859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; those disclosed in English et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P.Ed. Wiley - VCH, 2008; and those disclosed in Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289 - 302, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993. The entire contents of the above are hereby incorporated by reference into this specification.

[0378] In certain embodiments, the modified nucleobase is a nucleobase that has a structure that is fairly similar to the parent nucleobase, such as, for example, 7 - deazapurine, 5 - methylcytosine, or a G - clamp. In certain embodiments, the nucleobase mimetic includes a more complex structure, such as, for example, a tricyclic phenoxazine nucleobase mimetic. Methods for making the above - mentioned modified nucleobases are well - known to those skilled in the art.

[0379] Nucleic acid modification (sugar) The oligonucleotides provided herein can comprise 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 having a modified sugar moiety. For example, the furanosyl sugar ring of a nucleoside can be modified in a variety of ways including addition of substituents, cross-linking of two non-geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid, but not limited thereto. In certain embodiments, the oligonucleotide comprises 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

[0380] In certain embodiments of locked nucleic acids, 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 -O-N(R1)-,-C(R1)=C(R2)-O-,-C(R1)=N-,-C(R1)=N-O-,-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)- and is attached at the 4'-position by a linker selected from; where: x is 0, 1 or 2; n is 1, 2, 3 or 4; Each R1 and R2 is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, 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, a heterocyclic radical, a substituted heterocyclic radical, C1-C 12 aminoalkyl, substituted C1-C 12 aminoalkyl or a protecting group.

[0381] In certain embodiments, each of the linkers of the LNA compounds is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]n-O-, -C(R1R2)-N(R1)-O- or -C(R1R2)-O-N(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-O-N(R1)-2’ and 4’-CH2-N(R1)-O-2’-, where each R1 is independently H, a protecting group or C1-C 12 alkyl.

[0382] Certain LNAs have been manufactured and disclosed in 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. U.S.A., 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; Published U.S. patents and published applications disclosing LNAs include, for example, U.S. Patent 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publications 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0383] LNA is also provided herein in which the 2'-hydroxyl group of the ribosyl sugar ring is attached to the 4'-carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') linkage 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. Pat. Nos. 6,268,490 and 6,670,461). The linkage may be a methylene (-CH2- group bridging the 2'-oxygen atom and the 4'-carbon atom, where the term methyleneoxy (4'-CH2-O-2') LNA is used for the bicyclic moiety; when this position is an ethylene group, 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 very high duplex thermal stability (Tm = +3 to +10 °C), stability to 3'-exonuclease digestion, and good solubility properties with complementary DNA and RNA. Potent and non-toxic antisense oligonucleotides containing BNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

[0384] The 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 excellent stability against 3’-exonuclease. Alpha-L-methyleneoxy(4’-CH2-O-2’)LNA’ has been incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0385] The synthesis and production of methyleneoxy(4’-CH2-O-2’)LNA monomers of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil have been described together with oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The BNA and its production are also described in WO98 / 39352 and WO99 / 14226.

[0386] Analogs of methyleneoxy(4’-CH2-O-2’)LNA, phosphorothioate-methyleneoxy(4’-CH2-O-2’)LNA and 2’-thio-LNA have also been produced (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The production 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 2’-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog, has been described in the literature (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2’-amino- and 2’-methylamino-LNA have been produced and the thermal stability of duplexes with complementary RNA and DNA strands has been previously reported.

[0387] Modified sugar moieties are well known and can be used for modifying the affinity of an antisense compound to its target, typically increasing and / or increasing 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’ bridging) ENA; substituted sugars, especially 2’-substituted sugars having 2’-F, 2’-OCH3 or 2’-O(CH2)2-OCH3 substituents; and bicyclic modified sugars including 4’-thio modified sugars. The sugar can also be replaced, among other things, with a sugar mimetic group. Methods for producing modified sugars are well known to those skilled in the art. Some representative patents and publications teaching the production of such modified sugars include, but are not limited to, U.S. Pat. Nos. 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; 5,591,722; 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.

[0388] 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(CH2CH2O) n CH2CH2OR, n = 1 - 50; "locked" nucleic acids (LNA) in which the furanose moiety 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 amine (n = 1 - 10, amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2, among others.

[0389] The "deoxy" modification is hydrogen (i.e., deoxyribose sugar, particularly relevant to single-stranded overhangs); halo (e.g., fluoro); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) 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, for example, alkenyl and alkynyl optionally substituted with an amino functional group.

[0390] Other suitable 2'-modifications, such as modified MOE, are described in U.S. Patent Application Publication 20130130378, the content of which is incorporated herein by reference.

[0391] The modification at the 2'-position may be present in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent at C2' of ribose in the same configuration as at the 2'-OH of arabinose.

[0392] The sugar may contain two different modifications, e.g., gem modifications, at the same carbon of the sugar. The sugar moiety may also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon of ribose. Thus, the oligonucleotide may contain one or more monomers that include, for example, arabinose as the sugar. The monomer may have an alpha bond, e.g., an alpha - nucleoside, at the 1'-position of the sugar. The monomer may also have an opposite configuration at the 4'-position, e.g., C5' and H4' or substituents replacing them are interchanged with each other. When C5' and H4' or substituents replacing them are interchanged with each other, the sugar is said to be modified at the 4'-position.

[0393] The oligonucleotides disclosed herein also include abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having another chemical group in place of the nucleobase at C1'. See, e.g., U.S. Patent 5,998,203, which is incorporated herein by reference in its entirety. These abasic sugars may further include additional modifications to one or more of the constituent sugar atoms. The oligonucleotides may also include one or more sugars that are L-isomers, e.g., L-nucleosides. Modifications of the sugar moiety may also include replacement of 4'-O with sulfur, nitrogen optionally substituted, or a CH2 group. In certain embodiments, the linkage between C1' and the nucleobase is in the α configuration.

[0394] Sugar modifications may also include "acyclic nucleotides," which refer to any nucleotide having an acyclic ribose sugar, e.g., where the C-C bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') are absent, and / or where at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4') is independently or in combination absent from the nucleotide. In certain embodiments, the acyclic nucleotide is

Chemical formula

[0395] 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 having an arabinose configuration of 2'-O-Me.

[0396] When a particular nucleotide binds to an adjacent nucleotide at the 2'-position, it is understood that the sugar modification described herein can be replaced at the 3'-position of the sugar of the nucleotide to which it binds, for example, via the 2'-position. Modifications at the 3'-position are present in the xylose configuration. The term "xylose configuration" refers to the placement in the same configuration as the 3'-OH of the xylose sugar of the C3' substituent of ribose.

[0397] The hydrogen attached to C4' and / or C1' may be replaced with a linear or branched alkyl optionally substituted, alkenyl optionally substituted, or alkynyl optionally substituted, where the backbone of the alkyl, alkenyl, and alkynyl may include 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, aryl optionally substituted, heteroaryl optionally substituted, heterocyclic optionally substituted, or cycloalkyl optionally substituted, where R' is hydrogen, acyl, or aliphatic optionally substituted, and Z' is OR 11 、COR 11 、CO2R 11 、

Chemical formula

[0398] In certain embodiments, C4’ and C5’ together form an optionally substituted heterocyclic, 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 in each case an alkali metal or transition metal with an overall charge of +1; and Y is O, S or NR’, where R’ is hydrogen, optionally substituted aliphatic. Preferably this modification is at the 5’-end of the iRNA.

[0399] In certain embodiments, the oligonucleotide comprises at least two regions of at least two consecutive monomers of the above formula. In certain embodiments, the oligonucleotide comprises a gapped motif. In certain embodiments, the oligonucleotide comprises at least one region of about 8 to about 14 consecutive β-D-2’-deoxyribofuranosyl nucleosides. In certain embodiments, the oligonucleotide comprises at least one region of about 9 to about 12 consecutive β-D-2’-deoxyribofuranosyl nucleosides.

[0400] In certain embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) of the formula:

Chemical formula

[0401] In certain embodiments, the monomers include sugar mimetics. In some such embodiments, the mimetic is used in place of a sugar or sugar-nucleoside linkage combination, and the nucleobase maintains 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-nucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNA) linked by uncharged achiral linkages and morpholino groups. In some cases, 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 Acid Res. 2000, 28: 2911-14, incorporated herein by reference). Methods for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those of skill in the art.

[0402] Nucleic acid modification (sugar linkage) Linking groups are described herein for linking monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) to each other to thereby form 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 linking groups include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-).

[0403] As described above, [cyclic disulfide moiety] is described as being introduced into one or more of the phosphorus-containing nucleotide internucleotide linkages of an oligonucleotide as a temporary protecting group. The remaining phosphorus-containing nucleotide internucleotide linkages can also be modified using the methods described below.

[0404] Modified linkages can be used to modify, typically increase, the nuclease resistance of an oligonucleotide as compared to a native phosphodiester linkage. In certain embodiments, a linkage having a chiral atom can be produced as a racemic mixture, separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for making phosphorus-containing and non-phosphorus-containing linkages are well known to those of skill in the art.

[0405] The phosphate group in the linking group can be modified by replacement of one of the oxygen atoms with a different substituent. One result of this modification is an increase in the resistance of the oligonucleotide to nuclease degradation. Examples of modified phosphate groups include phosphorothioates, phosphorosenates, boranophosphates, borano phosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In certain embodiments, one of the non-bridging phosphate oxygen atoms in the linkage can be replaced with any of the following: S, Se, BR3 (where R is hydrogen, alkyl, aryl), C (i.e., an alkyl group, aryl group, etc.), H, NR2 (where R is hydrogen, optionally substituted alkyl, aryl) or (where R is optionally substituted alkyl or aryl). The phosphorus atom of an unmodified phosphate group is achiral. However, replacement of one of the above atoms or groups of atoms of the non-bridging oxygen results in the phosphorus atom becoming chiral; in other words, the phosphorus atom in the phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can have either an "R" configuration (here Rp) or an "S" configuration (here Sp).

[0406] Phosphorothioates have both non-bridging oxygens replaced by sulfur. The phosphorus center of phosphorothioates is achiral, making the formation of oligonucleotide diastereomers impossible. Thus, without wishing to be bound by theory, modifications to both non-bridging oxygens that eliminate a chiral center, such as phosphorothioate formation, may be desirable as they cannot form a mixture of diastereomers. Thus, the non-bridging oxygen can independently be one of O, S, Se, B, C, H, N or OR (where R is alkyl or aryl).

[0407] Phosphate linkers can also be modified by the exchange of the bridging oxygen (i.e., the oxygen that binds the phosphate to the sugar of the monomer) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylenephosphonate). The replacement can occur at one or both of the linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, replacement with nitrogen is preferred.

[0408] A modified phosphate bond in which at least one of the oxygens binding to the phosphate is replaced or the phosphate group is replaced by a non-phosphorus group can also be referred to as a "non-phosphodiester sugar-sugar bond" or a "non-phosphodiester linker".

[0409] In certain 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, since the charged phosphodiester group is the reaction center for nucleic acid degradation, the exchange with its neutral structural mimetic is thought to increase nuclease stability. Similarly, without wishing to be bound by theory, in certain embodiments, the introduction of a change in which the charged phosphate group is replaced by a neutral moiety may be desirable.

[0410] Examples of moieties that can replace the phosphate group include amides (e.g., amide-3 (3’-CH2-C(=O)-N(H)-5’) and amide-4 (3’-CH2-N(H)-C(=O)-5’)), hydroxylamino, siloxanes (dialkylsiloxanes), carboxamides, carbonates, carboxymethyl, carbamates, carboxylic acid esters, thioethers, ethylene oxide linkers, sulfides, sulfonates, sulfonamides, sulfonate esters, thioformacetal (3’-S-CH2-O-5’), formacetal (3’-O-CH2-O-5’), oximes, methyleneimino, methylenecarbonylamino, methylenemethylimino (MMI, 3’-CH2-N(CH3)-O-5’), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamide (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’, C3’-CH2-NH-NH-C5’, 3’-NHP(O)(OCH3)-O-5’ and 3’-NHP(O)(OCH3)-O-5’ and non-ionic bonds containing mixed N, O, S and CH2 moieties, but are not limited thereto. See, for example, Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amides, carbamates and ethylene oxide linkers.

[0411] One of ordinary skill in the art will fully recognize that in some instances, the exchange of non-bridging oxygen can lead to cleavage of the sugar-sugar bond by the neighboring 2’-OH, and thus in many cases, modification of the non-bridging oxygen requires modification of the 2’-OH, e.g., modification that does not participate in cleavage of the neighboring sugar-sugar bond, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA.

[0412] Preferred non-phosphodiester sugar linkages include phosphorothioates, phosphorothioates with at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Sp isomer, phosphorothioates with at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonate), selenophosphates, phosphoramidates (e.g., N-alkyl phosphoramidates) and boranophosphonates.

[0413] In certain embodiments, the oligonucleotide further comprises at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and including all up to the maximum) modification or non-phosphodiester bond. In certain embodiments, the oligonucleotide further comprises at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and including all up to the maximum) phosphorothioate bond.

[0414] Oligonucleotides can also be constructed in which the phosphate linker and sugar are replaced with nuclease-resistant nucleosides or nucleotide surrogates. Without wishing to be bound by theory, the absence of a charged repeating backbone is thought to reduce binding to proteins that recognize polyanions (e.g., nucleases). Similarly, without wishing to be bound by theory, in certain embodiments, it may be desirable to introduce modifications in which the bases are tethered by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acids (PNA), aminoethylglycyl PNA (aegPNA) and backbone extended pyrrolidine PNA (bepPNA) nucleoside surrogates. Preferred surrogates are PNA surrogates.

[0415] The oligonucleotides described herein may contain one or more asymmetric centers and thus give rise to other stereoisomeric arrangements such as may be defined as (R) or (S) for sugar anomers or (D) or (L) for amino acids in terms of enantiomers, diastereomers, and absolute stereochemistry. Included in the oligonucleotides are all such possible isomers as well as their racemic and optically pure forms.

[0416] Nucleic acid modification (terminal 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).

[0417] In certain embodiments, the 5' end of the antisense strand does not comprise 5'-vinylphosphonate (VP).

[0418] The ends of the iRNA agent can be modified. Such modifications can be at one or both ends. For example, the 3' and / or 5' ends of the iRNA can be conjugated to other functional molecular entities such as a labeling moiety, e.g., a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or a protecting group (e.g., based on sulfur, silicon, boron or esters). The functional molecular entity can be attached to the sugar via a phosphate group and / or a linker. The terminal atom of the linker can be attached 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 attached to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

[0419] When a linker / phosphate functional molecular entity-linker / phosphate array is inserted between the two strands of a double-stranded oligonucleotide, this array can be an alternative to the hairpin loop in a hairpin-type oligonucleotide.

[0420] Terminal modifications useful for activity modulation include modification of the 5' end of the iRNA with a phosphate or phosphate analog. In certain embodiments, the 5' end of the iRNA is phosphorylated or includes a phosphoril analog. Examples of 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Modifications at the 5' end may also be useful for stimulating or inhibiting the immune system of a subject. In certain embodiments, the 5' end of the oligonucleotide is modified

Chemical formula

[0421] Examples of 5' modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); 5'-alpha-thiotriphosphate; 5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoroamidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'). Other 5' modifications include 5'-alkylphosphonates (R(OH)(O)P-O-5', R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)P-O-5', R = alkyl ether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc.); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp) and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'). Examples of other 5' modifications include, here, where Z is alkyl optionally substituted at least once, e.g., ((HO)2(X)P-O[-(CH2) a -O-P(X)(OH)-O] b -5’, ((HO)2(X)P-O[-(CH2) a -P(X)(OH)-O] b -5’, ((HO)2(X)P-[-(CH2) a -O-P(X)(OH)-O] b -5’; dialkyl terminal phosphates and phosphate mimics: HO[-(CH2) a -O-P(X)(OH)-O] b -5’, H2N[-(CH2) a-O-P(X)(OH)-O] b -5’, H[-(CH2) a -O-P(X)(OH)-O] b -5’, Me2N[-(CH2) a -O-P(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 -including -5’, where a and b are each independently 1 to 10. Other embodiments include the exchange of oxygen and / or sulfur with BH3, BH3 - and / or Se.

[0422] Terminal modifications can also be useful for monitoring the distribution, in which case preferred groups to be added include fluorophores such as fluorescein or Alexa dyes such as Alexa 488. Terminal modifications can also be useful for enhancing uptake, and useful modifications for this purpose include targeting ligands. Terminal modifications can also be useful for cross-linking oligonucleotides to other moieties; useful modifications for this purpose include mitomycin C, psoralen and their derivatives.

[0423] Destabilizing modification Oligonucleotides such as iRNA or dsRNA agents can be optimized for RNA interference by introducing destabilizing modifications at sites opposite to the seed region (i.e., positions 2 to 8 at the 5’ end of the antisense strand) of the sense strand or antisense strand of the iRNA duplex, thereby increasing the tendency of the iRNA duplex to dissociate or melt (decrease in free energy of duplex binding). This modification can increase the tendency of the duplex to dissociate or melt in the seed region of the antisense strand.

[0424] The destabilizing modifications include abasic modifications; mismatches with opposite nucleotides in the opposite strand; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides such as unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA).

[0425] Examples of abasic modifications are:

Chem.

[0426] Examples of sugar modifications are:

Chem.

[0427] The term "UNA" refers to unlocked acyclic nucleic acids in which any of the sugar linkages have been removed to form unlocked "sugar" residues. In one example, UNA also includes monomers in which the C1'-C4' linkage (i.e., the shared carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In other examples, the C2'-C3' linkage of the sugar (i.e., the shared carbon-carbon bond between the C2' and C3' carbons) is 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). The acyclic derivatives provide a high degree of backbone flexibility without affecting Watson-Crick base pairing. The acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0428] The term "GNA" is a glycol nucleic acid, a polymer similar to DNA or RNA but different in that its "backbone" consists of repeating glycerol units linked by phosphodiester bonds.

Chem.

[0429] The destabilizing modification is a mismatch (i.e., a non-complementary base pair) between the destabilizing nucleotide of the dsRNA duplex and the nucleotide of the reverse strand. Examples of mismatched 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 mismatched base pairs known in the art are also applicable in the present invention. The mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatched base pairing can occur between the nucleobases from each nucleotide, independent of the modification of the ribose sugar of the nucleotide. In certain embodiments, an oligonucleotide such as an siRNA or an iRNA agent includes at least one nucleobase in a mismatched pairing that is a 2'-deoxy nucleobase; for example, the 2'-deoxy nucleobase is in the sense strand.

[0430] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0431] The destabilizing modification can also include universal bases with phosphate modifications that have a reduced or eliminated ability to form hydrogen bonds with reverse bases.

[0432] In certain embodiments, the antisense strand includes one destabilizing nucleotide that is not a terminal nucleotide and not a cleavage region nucleotide. Optionally, the destabilizing nucleotide is glycol nucleic acid (GNA). Optionally, the destabilizing nucleotide is unlocked nucleic acid (UNA). Optionally, the destabilizing nucleotide is a 2'-5' linked ribonucleotide (3'-RNA).

[0433] Further examples of destabilizing nucleotides are [Table 36] comprising the same or a stereoisomer thereof, wherein B is a modified or unmodified nucleobase and the asterisk represents R, S or racemic (e.g., S-GNA). In other embodiments, the destabilizing nucleotide comprises a nucleobase mismatch between the antisense strand and the sense strand; for example, the sense strand may have a mismatch with the antisense strand while the latter maintains a match with the target mRNA at the same position.

[0434] Nucleobase modifications that impair or completely abolish the ability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilization of the central region of the sRNA duplex as described in WO2010 / 0011895, which is hereby incorporated by reference in its entirety. Examples of nucleobase modifications are:

Chemical formula

[0435] Examples of phosphate modifications that are known to reduce the thermal stability of the dsRNA duplex compared to the native phosphodiester bond are:

Chemical formula

[0436] In certain embodiments, the oligonucleotide may comprise 2'-5' linkages (having 2'-H, 2'-OH and 2'-OMe and having P=O or P=S). For example, 2'-5' linkage modifications can be used at the 5' end of the sense strand to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand or to avoid activation of the sense strand by RISC.

[0437] In other embodiments, the oligonucleotide is an L sugar (e.g., L-ribose, L-arabinose having 2'-H, 2'-OH and 2'-OMe). For example, these L sugar modifications can be used at the 5' end of the sense strand to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand or to avoid activation of the sense strand by RISC.

[0438] In certain embodiments, one or more targeting ligands are optionally attached to the modified phosphate prodrug compound via one or more linkers / tethers and via any of R2, R3, R4, R5, R6, R7, R8, and R9 of the [cyclic disulfide moiety].

[0439] The introduction of targeting ligands to the oligonucleotide via the [cyclic disulfide moiety] to the sense strand or the antisense strand or both the sense strand and the antisense strand is described in Scheme 16 of Example 10 below. These targeting ligands can be cleaved together with the [cyclic disulfide moiety] after the siRNA oligonucleotide enters the cytosol.

[0440] In certain 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, a folate, a thyrotropin, a melanotropin, surfactant protein A, a mucin, a glycosylated polyamino acid, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipophilic moiety that enhances plasma protein binding, cholesterol, a steroid, a bile acid, vitamin B12, biotin, a fluorophore, and a peptide.

[0441] In certain embodiments, at least one ligand is a carbohydrate-based ligand that targets liver tissue. In certain embodiments, the carbohydrate-based ligand is selected from the group consisting of galactose, polyvalent galactose, N-acetyl-galactosamine (GalNAc), polyvalent GalNAc, mannose, polyvalent mannose, lactose, polyvalent lactose, N-acetyl-glucosamine (GlcNAc), polyvalent GlcNAc, glucose, polyvalent glucose, fucose, and polyvalent fucose.

[0442] In certain embodiments, at least one ligand is a lipophilic moiety. In certain embodiments, the lipophilicity of the lipophilic moiety is greater than 0 as measured by logK ow or the hydrophobicity of the compound is greater than 0.2 as measured by the unbound fraction in the plasma protein binding assay of the compound.

[0443] In certain embodiments, the lipophilic moiety is a saturated or unsaturated C4-C 30 hydrocarbon chain and includes an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety includes a saturated or unsaturated C6-C 18 hydrocarbon chain.

[0444] Further details regarding the lipophilicity and hydrophobicity of additional lipophilic moieties and of the lipophilic moiety of the oligonucleotide can be found in PCT Application PCT / US20 / 59399, entitled "Extrahepatic Delivery", filed on November 6, 2020, the content of which is hereby incorporated by reference in its entirety.

[0445] In certain embodiments, at least one ligand targets a receptor involved in delivery to the CNS tissue. In certain 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.

[0446] In certain embodiments, at least one ligand targets a receptor involved in delivery to the eye tissue. In certain embodiments, the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate-based ligand.

[0447] The targeting ligand can also be introduced directly into the oligonucleotide (independently, i.e., not via the [cyclic disulfide moiety]).

[0448] In certain embodiments, the oligonucleotide comprises at least one targeting ligand at the 5'-end, 3'-end, and / or internal position of the antisense strand.

[0449] In certain embodiments, the oligonucleotide comprises at least one targeting ligand at the 5'-end, 3'-end, and / or internal position of the sense strand.

[0450] In certain embodiments, 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.

[0451] 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.

[0452] In certain embodiments, one or more targeting ligands are attached to the modified phosphate prodrug compound (via the [cyclic disulfide moiety]) via one or more linkers / tethers as follows.

[0453] In certain embodiments, one or more targeting ligands are attached directly to the oligonucleotide (i.e., not via the [cyclic disulfide moiety]) via one or more linkers / tethers as follows.

[0454] Linker / tether The linker / tether is attached to the modified phosphate prodrug compound at the "tethering attachment point (TAP)". The linker / tether can be any C1-C 100 carbon-containing moiety (e.g., C1-C75 , C1-C 50 , C1-C 20 , C1-C 10 ; C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 ) may be included and may contain at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amide (NHC(O)-) group of a linker / tether that can serve as a point of attachment for the modified phosphate prodrug compound. Non-limiting examples of the linker / tether (underlined) are 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-; TAP- 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)-including; where n is from 1 to 20 (for example, 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, nitrogen may form part of a terminal oxyamino group, for example, -ONH2 or a hydrazino group, -NHNH2. The linker / tether may optionally be substituted, for example, with hydroxy, alkoxy, perhaloalkyl and / or may optionally have one or more additional heteroatoms, for example, N, O or S inserted. Preferred tethering ligands are, 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); TAP- C(O)-O(ligand) ; TAP- C(O)-(CH2 ) 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) ; Or TAP- (CH 2 ) n -NH-C(O)(ligand) may include. In certain embodiments, the amino-terminal linker / tether (e.g., NH2, ONH2, NH2NH2) can form an imino bond (i.e., C=N) with the ligand. In certain embodiments, the amino-terminal linker / tether (e.g., NH2, ONH2, NH2NH2) can be acylated with, for example, C(O)CF3.

[0455] In certain embodiments, the linker / tether can be terminated with a mercapto group (i.e., SH) or an olefin (e.g., CH=CH2). 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 )and can be, where n can be as described elsewhere. The tether can optionally be substituted, for example, with hydroxy, alkoxy, perhaloalkyl and / or optionally have one or more additional heteroatoms inserted, for example, N, O or S. The double bond can be cis or trans or E or Z.

[0456] In other embodiments, the linker / tether preferably includes an electrophilic moiety at the terminal position of the linker / tether. Examples of electrophilic moieties are, for example, aldehyde, alkyl halide, mesylate, tosylate, nosylate or brosylate or activated carboxylic acid esters, such as NHS ester or a pentafluorophenyl ester. Preferred linkers / tethers (underlined) are TAP-(CH2) n CHO; TAP-C(O)(CH2) n CHO; or TAP-NR’’’’(CH2) n CHO (where n is from 1 to 6 and R’’’’ is C1-C6 alkyl); or TAP-(CH2) n C(O)ONHS; TAP-C(O)(CH2) n C(O)ONHS; or TAP-NR’’’’(CH2) n C(O)ONHS (where n is from 1 to 6 and R’’’’ is C1-C6 alkyl); TAP-(CH2) n C(O)OC6F5; TAP-C(O)(CH2) n C(O)OC6F5; or TAP-NR’’’’(CH2) n C(O)OC6F5 (where n is from 1 to 11 and R’’’’ is C1-C6 alkyl); or -(CH2) n CH2LG; TAP-C(O)(CH2) n CH2LG; or TAP-NR’’’’(CH2) nCH2LG (wherein n may be as described elsewhere in this specification and R’’’’ is C1-C6 alkyl), where LG may be a leaving group such as a halide, mesylate, tosylate, nosylate, brosylate. The tethering is by coupling of a nucleophilic group of the ligand such as a thiol or amino group with an electrophilic group of the tether.

[0457] In other embodiments, it may be desirable for the monomer to include a phthalimide group (K) at the terminal position of the linker / tether.

Chemical formula

[0458] In other embodiments, other protected amino groups may be at the terminal position of the linker / tether such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc or arylsulfonyl (for example, the aryl moiety may be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0459] Any of the linkers / tethers described herein may further include one or more additional linking groups such as -O-(CH2) n -, -(CH2) n -SS-, -(CH2) n - or -(CH=CH)-.

[0460] Cleavable linker / tether In certain embodiments, at least one of the linkers / tethers may be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker or a peptidase-cleavable linker.

[0461] In certain embodiments, at least one of the linkers / tethers may be a linker cleavable by reduction (such as a disulfide group).

[0462] In certain embodiments, at least one of the linker / tether can be an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group).

[0463] In certain embodiments, at least one of the linker / tether can be an esterase-cleavable linker (e.g., an ester group).

[0464] In certain embodiments, at least one of the linker / tether can be a phosphatase-cleavable linker (e.g., a phosphate group).

[0465] In certain embodiments, at least one of the linker / tether can be a peptidase-cleavable linker (e.g., a peptide bond).

[0466] The cleavable linking group is sensitive to a cleaving agent, e.g., the presence of pH, redox potential, or a degradable molecule. Generally, the cleaving agent is present or has a higher level or activity intracellularly than in serum or blood. Examples of such degrading agents include: for example, oxidizing or reducing enzymes or reducing agents present in cells, such as mercaptans, that can degrade a redox-cleavable linking group by oxidation or reduction; esterases; agents that create an endosomal or acidic environment, e.g., agents that result in a pH of 5 or less; general acids, peptidases (which can be substrate-specific), and enzymes that can hydrolyze or degrade an acid-cleavable linking group by acting as phosphatases.

[0467] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, in the range of about 7.1 - 7.3. Endosomes have a more acidic pH, in the range of 5.5 - 6.0, and lysosomes have an even more acidic pH of about 5.0. Some tethers have a linking group that cleaves at a preferred pH, thereby releasing the iRNA agent within the cell or a desired cellular compartment to a ligand (e.g., a targeting or cell-permeable ligand, such as cholesterol).

[0468] The chemical conjugation (e.g., linker) that binds the ligand to the iRNA agent may include a disulfide bond. When the iRNA agent / ligand complex is taken up by cells via endocytosis, the acidic environment of the endosome cleaves the disulfide bond, 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.

[0469] The tether contains a linker that is cleavable by a specific enzyme. The type of linker incorporated into the tether can depend on the cell targeted by the iRNA agent. For example, an iRNA agent targeting hepatocyte mRNA can be conjugated with a tether containing an ester group. Hepatocytes are rich in esterase, and thus the tether is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase. Cleavage of the tether can release 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 esterase include cells of the lung, renal cortex, and testis.

[0470] A tether containing a peptide bond can be conjugated to an iRNA agent targeting cell types rich in peptidase, such as hepatocytes and synoviocytes. For example, an iRNA agent targeting synoviocytes, such as for the treatment of an inflammatory disease (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond.

[0471] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a cleaving agent (or condition) to cleave the candidate linker. It may also be desirable to test the ability of the candidate cleavable linker to withstand cleavage when in blood or when in contact with other non-target tissues, such as tissues that are exposed to the iRNA agent when administered to a subject. Thus, determine the relative sensitivity to cleavage between a first and a second condition, where the first is selected to be an indicator of cleavage within the target cell and the second is selected to be an indicator of cleavage in other tissues or body fluids, such as blood or serum. The 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 an initial evaluation under cell-free or culture conditions and confirm with a further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is 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) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0472] Redox cleavable linker A class of cleavable linkers are redox-cleavable linkers that are cleaved by reduction or oxidation. An example of a linker cleavable by reduction is a disulfide linker (-S-S-). The methods described herein may be considered to determine whether a candidate cleavable linker is a suitable "reduction-cleavable linker" or, for example, suitable for use with a particular iRNA moiety and a particular targeting agent. For example, a candidate can be evaluated by incubating it with a reducing agent that mimics the cleavage rate observed in cells, e.g., dithiothreitol (DTT) or other reagents known in the art. A candidate can also be evaluated under conditions that mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved up to 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2-fold, 4-fold, 10-fold or 100-fold faster in cells (or in vitro conditions selected to mimic intracellular conditions) compared to blood (or in vitro conditions selected to mimic extracellular conditions). The cleavage rate of a candidate compound can be determined using a standard enzyme reaction rate assay under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0473] Phosphate-based cleavable linker Phosphate-based linking groups are cleaved by agents that decompose or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0474] Acid-cleavable linking group An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved by an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or an agent such as an enzyme that can act as a general acid. In cells, specific low-pH organelles such as endosomes and lysosomes can provide the cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. The acid-cleavable group can have the general formula -C=NN-, C(O)O or -OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using a method similar to that described above.

[0475] Ester-based linking group Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. The ester-cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using a method similar to that described above.

[0476] Peptide-based C-leaving group Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids that give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not contain an amide group. The amide group can be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids that gives rise to peptides and proteins. Peptide-based cleavage 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 functional group. The peptide cleavable linking group has 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 a method similar to that described above.

[0477] Biodegradable linker / tether The linker may also include a biodegradable linker that is a nucleotide and non-nucleotide linker or a combination thereof that connects two portions of a molecule, e.g., one or both ends of two individual siRNA molecules, to produce bis(siRNA). In certain embodiments, mere electrostatic or stacking interactions between two individual siRNAs can represent the linker. Non-nucleotide linkers can be tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides and their derivatives, aliphatic, cycloaliphatic, heterocyclic and combinations thereof.

[0478] In certain embodiments, at least one of the linkers (tethers) is a biodegradable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose and mannose and combinations thereof.

[0479] In certain embodiments, the biodegradable carbohydrate linker may have from 1 to 10 saccharide units having at least one anomeric bond connecting two siRNA units. When two or more saccharides are present, these units may be linked via 1-3, 1-4 or 1-6 glycosidic bonds or alkyl chains.

[0480] Examples of biodegradable linkers include:

Chem.

Chem.

Chem.

[0481] Further description of the biodegradable linker can be found in PCT application PCT / US18 / 14213 entitled "Endosomal Cleavable Linkers" filed on January 18, 2018, the contents of which are hereby incorporated by reference in their entirety.

[0482] Carrier In certain embodiments, one or more targeting ligands may be attached to the modified phosphorous prodrug compound (via the [cyclic disulfide moiety]), via one or more carriers described herein, and optionally via one or more of the linkers / tethers described above.

[0483] In certain embodiments, one or more targeting ligands may be attached directly to the oligonucleotide (i.e., without the [cyclic disulfide moiety]), via one or more carriers described herein, and optionally via one or more of the linkers / tethers described above.

[0484] The carrier can be a cyclic group or an acyclic group. In certain 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 certain embodiments, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0485] The carrier can replace one or more nucleotides of the iRNA agent.

[0486] In certain embodiments, the carrier replaces one or more nucleotides at an internal position of the iRNA agent.

[0487] In other embodiments, the carrier replaces nucleotides at the end of the sense strand or the antisense strand. In certain embodiments, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby functioning as an end cap that protects the 3' end of the sense strand. In certain embodiments, the carrier can be a cyclic group having an amine, for example, the carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0488] A ribonucleotide subunit in which the ribose sugar of the subunit is replaced is herein referred to as a ribose-exchanged modified subunit (RRMS). The carrier can be a cyclic or acyclic moiety and includes two "backbone attachment points" (e.g., hydroxyl groups) and a ligand. The targeting ligand can bind directly to the carrier or can bind indirectly to the carrier via the intervening linker / tether.

Chemical Structure

[0489] The ligand-conjugate monomer subunit may be a 5' or 3' terminal subunit of the iRNA molecule, i.e., one of the two "W" groups may be a hydroxyl group and the other "W" group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugate monomer subunit may occupy an internal position and both "W" groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugate monomer subunit may be present in the iRNA agent.

[0490] Sugar-exchange-based monomers, e.g., ligand-conjugate monomers (cyclic) Cyclic sugar-exchange based monomers, e.g., sugar-exchange based ligand-conjugate monomers, are also referred to herein as RRMS monomer compounds. The carrier has the general formula (LCM-2) provided below (in its structure, the preferred backbone attachment points are where Y is CR 9 R 10 if so then R 1 or R 2 ; R 3 or R 4 ; or R 9 and R 10 selected from (the two positions are selected such that they are two backbone attachment points, e.g., R 1 and R 4 or R 4 and R 9 ). The preferred tethering attachment points include R 7 ; R 5 or R 6 when X is CH2. The carrier is described below as an entity that can be incorporated into a chain. Thus, the structure also includes one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R 1 or R 2 ; R 3 or R 4 ; or R 9 or R 10 (where Y is CR 9 R 10It is understood that when it is so, it includes the situation where it is bonded to phosphate or modified phosphate, for example, bonded to a sulfur-containing main chain. For example, one of the above R groups is -CH2-, where one bond is bonded to the carrier and one is bonded to a main chain atom, for example, a linking oxygen or a central phosphorus atom.

Chemical formula

[0491] Examples of carriers are, for example, where 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 and Z is CR 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 and Z is CR 7 or NR 7 and Y is O and Z is CR 11 R 12 or 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 C6 cycloalkyl (H, z = 2) or indane ring system, for example, 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).

[0492] In certain embodiments, the carrier may be based on a pyrroline ring system or a 4-hydroxyproline ring system, for example, X is N(CO)R 7 or NR 7 and Y is CR 9R 10 is present, and Z is absent (D). [Chemical formula] OFG 1 is preferably a primary carbon attached to one of the carbons of a 5-membered ring, for example, an exocyclic alkylene group, for example, a methylene group attached to -CH2OFG in D 1 ). OFG 2 is preferably directly attached to one of the carbons of a 5-membered ring (-OFG in D 2 ). For a pyrroline-based carrier, -CH2OFG 1 may be attached to C-2, and OFG 2 may be attached at C-3; or -CH2OFG 1 may be attached to C-3, and OFG 2 may be attached to C-4. In certain embodiments, CH2OFG 1 and OFG 2 may be geminally substituted on one of the above carbons. For a 3-hydroxyproline-based carrier, -CH2OFG 1 may be attached to C-2, and OFG 2 may be attached to C-4. Thus, pyrroline- and 4-hydroxyproline-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted by limitations, for example, due to the presence of a ring, for a particular bond. Thus, CH2OFG 1 and OFG 2 can be cis or trans to each other in any of the above conformations. Accordingly, all cis / trans isomers are explicitly included. The monomers may also contain one or more chiral centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are explicitly included (e.g., CH2OFG 1 and OFG 2The centers having them may both have an R configuration; or both have an S configuration; or one center may have an R configuration and the other center may have an S configuration, and vice versa). The tethering junction point is preferably nitrogen. Preferred examples of the carrier D include the following. [Chemical formula]

[0493] In certain embodiments, the carrier may be based on a piperidine ring system (E), for example, X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 and is. [Chemical formula] OFG 1 is preferably a primary carbon bonded to one of the carbons in the six-membered ring, for example, an exocyclic alkylene group, for example, a methylene group (n = 1) or an ethylene group (n = 2) bonded to - (CH2) in E n OFG 1 . OFG 2 is preferably directly bonded to one of the carbons in the six-membered ring (-OFG in E 2 ). - (CH2) n OFG 1 and OFG 2 may be arranged in a geminal-like manner in the ring, that is, both groups may be bonded to the same carbon, for example, C-2, C-3 or C-4. Alternatively, - (CH2) n OFG 1 and OFG 2 may be arranged in a vicinal-like manner in the ring, that is, both groups may be bonded to adjacent ring carbon atoms, for example, - (CH2) n OFG 1 may be bonded to C-2, and OFG 2 may be bonded to C-3; - (CH2) n OFG 1 may be bonded to C-3, and OFG 2may 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. Thus, the piperidine-based monomer may include a bond (e.g., a carbon-carbon bond) where bond rotation is restricted for a particular bond, e.g., due to the presence of a ring. Thus, -(CH2) OFG n and OFG 1 and OFG 2 may be cis or trans to each other in any of the above pairings. Accordingly, the all-trans isomer is explicitly included. The monomer may also include one or more chiral centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomer are explicitly included (e.g., the centers having CH2OFG 1 and OFG 2 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 junction is preferably nitrogen.

[0494] 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 may be based on 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 12

Chemical formula

[0495] In certain embodiments, the carrier can be based on a decalin ring system, for example, X is CH2; Y is CR 9 R 10 ; Z is CR 11 R 12 , and R 5 and R 11combine to form a C6 cycloalkyl (H, z = 2) or an indane ring system. For example, X is CH2; Y is CR 9 R 10 ; Z is CR 11 R 12 ; and R 5 and R 11 combine to form a C5 cycloalkyl (H, z = 1).

Chemical formula

[0496] Other carriers may include those based on 3-hydroxyproline (J).

Chemical formula

[0497] Further details regarding representative cyclic, sugar-exchange-based carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are hereby incorporated by reference in their entirety.

[0498] Sugar-exchange-based monomers (acyclic) Acyclic sugar-exchange-based monomers, for example, sugar-exchange-based ligand-conjugate monomers, are also referred to herein as ribose-exchange monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers are of formula LCM-3 or LCM-4:

Chemical formula

[0499] In certain embodiments, each of x, y, and z can independently 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 (for example, based on serinol), x is 0, y and z are each 1, and in formula LCM-3, y and z are each 1. The following formula LCM-3 or LCM-4 can optionally be substituted, for example, with hydroxy, alkoxy, perhaloalkyl.

[0500] Further details regarding representative acyclic, sugar-exchange-based carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are hereby incorporated by reference in their entirety.

[0501] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated, optionally via a carrier and / or a linker / tether, to the 5'-end of the sense strand or the 5'-end of the antisense strand.

[0502] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated, optionally via a carrier and / or a linker / tether, to the 3'-end of the sense strand or the 3'-end of the antisense strand.

[0503] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated, optionally via a carrier and / or a linker / tether, to both ends of the sense strand.

[0504] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated, optionally via a carrier and / or a linker / tether, to both ends of the antisense strand.

[0505] In certain embodiments, the oligonucleotide comprises one or more targeting ligands conjugated, optionally via a carrier and / or a linker / tether, to an internal position of the sense strand or the antisense strand.

[0506] In certain embodiments, one or more targeting ligands are conjugated to the ribose, nucleobase and / or internucleotide linkage. In certain embodiments, one or more targeting ligands are conjugated to the ribose at the 2', 3', 4' and / or 5'-position of the ribose. In certain embodiments, one or more targeting ligands are conjugated to a natural (e.g., A, T, G, C or U) or a modified nucleobase as defined herein. In certain embodiments, one or more targeting ligands are conjugated to a phosphate or modified phosphate group as defined herein.

[0507] 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 of the same or different targeting ligands conjugated to the 5' or 3' end of the antisense strand.

[0508] In certain embodiments, at least one targeting ligand is located at one or more terminal positions of the sense or antisense strand. In certain embodiments, at least one targeting ligand is located at the 3' or 5' end of the sense strand. In certain embodiments, at least one targeting ligand is located at the 3' or 5' end of the antisense strand.

[0509] In certain embodiments, at least one targeting ligand conjugates to one or more internal positions of at least one strand. An internal position of a strand refers to a nucleotide at any position of the strand excluding the terminal positions from the 3' and 5' ends of the strand (e.g., excluding two positions: the 1st position counted from the 3' end and the 1st position counted from the 5' end).

[0510] In certain embodiments, at least one targeting ligand is located at one or more internal positions of at least one strand, which includes all positions excluding the two terminal positions from each end of the strand (e.g., excluding four positions: the 1st and 2nd positions counted from the 3' end and the 1st and 2nd positions counted from the 5' end). In certain embodiments, the targeting ligand is located at one or more internal positions of at least one strand, which includes all positions excluding the three terminal positions from each end of the strand (e.g., excluding six positions: the 1st, 2nd, and 3rd positions counted from the 3' end and the 1st, 2nd, and 3rd positions counted from the 5' end).

[0511] In certain embodiments, at least one targeting ligand is located at one or more positions at at least one end of the double-stranded region, which includes all positions within the double-stranded region but does not include the overhang region or a carrier that replaces the terminal nucleotide at the 3' end of the sense strand.

[0512] In certain embodiments, at least one targeting ligand is located in the sense strand within the first 5, 4, 3, 2, or 1 base pairs at the 5' end of the antisense strand of the double-stranded region.

[0513] In certain embodiments, at least one targeting ligand (e.g., a lipophilic moiety) is located at one or more internal positions of at least one strand, which, excluding the cleavage site region of the sense strand, for example, the targeting ligand (e.g., a lipophilic moiety) is not located at positions 9 - 12 counted from the 5' end of the sense strand, for example, the targeting ligand (e.g., a lipophilic moiety) is not located at positions 9 - 11 counted from the 5' end of the sense strand. Alternatively, the internal positions exclude positions 11 - 13 counted from the 3' end of the sense strand.

[0514] In certain embodiments, at least one targeting ligand (e.g., a lipophilic moiety) is located at one or more internal positions of at least one strand, which excludes the cleavage site region of the antisense strand. For example, the internal positions exclude positions 12 - 14 counted from the 5' end of the antisense strand.

[0515] In certain embodiments, at least one targeting ligand (e.g., a lipophilic moiety) is located at one or more internal positions of at least one strand, which excludes positions 11 - 13 of the sense strand counted from the 3' end and positions 12 - 14 of the antisense strand counted from the 5' end.

[0516] In certain embodiments, one or more targeting ligands (e.g., a lipophilic moiety) are located at one or more of the following internal positions: positions 4 - 8 and 13 - 18 of the sense strand and positions 6 - 10 and 15 - 18 of the antisense strand counted from the 5' end of each strand.

[0517] In one embodiment, one or more targeting ligands (e.g., a lipophilic moiety) are located at one or more of the following internal positions: positions 5, 6, 7, 15, and 17 of the sense strand and positions 15 and 17 of the antisense strand, counted from the 5' end of each strand.

[0518] Target gene Without limitation, target genes of siRNA include, but are not limited to, genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, genes that express kinases, adapter protein genes, genes encoding G protein superfamily molecules, genes encoding transcription factors, genes involved in angiogenesis, viral genes, genes essential for viral replication, cellular genes involved in viral function, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes involved in unwanted immune responses, genes involved in pain processing, genes involved in neurological diseases, and one allele gene of allelic genes or polymorphic genes found in cells characterized by heterozygosity.

[0519] Examples of specific 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; Cyclin D gene; VEGF gene; EGFR gene; Cyclin A gene; Cyclin E 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 gene, 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 gene, genes necessary for human papillomavirus replication, Human immunodeficiency virus gene, genes necessary for human immunodeficiency virus replication, Hepatitis A virus gene, genes necessary for hepatitis A virus replication, Hepatitis B virus gene, genes necessary for hepatitis B virus replication, Hepatitis C virus gene, genes necessary for hepatitis C virus replication, Hepatitis D virus gene, genes necessary for hepatitis D virus replication, Hepatitis E virus gene, genes necessary for hepatitis E virus replication, Hepatitis F virus gene, genes necessary for hepatitis F virus replication, Hepatitis G virus gene, genes necessary for hepatitis G virus replication, Hepatitis H virus gene, genes necessary for hepatitis H virus replication, Respiratory syncytial virus gene,Genes essential for the replication of respiratory syncytial virus, herpes simplex virus genes, genes essential for the replication of herpes simplex virus, cytomegalovirus genes, genes essential for the replication of cytomegalovirus, Epstein-Barr virus genes, genes essential for the replication of Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus genes, genes essential for the replication of Kaposi's sarcoma-associated herpesvirus, JC virus genes, human genes essential for the replication of JC virus, mumps virus genes, genes essential for the replication of mumps virus, rhinovirus genes, genes essential for the replication of rhinovirus, coronavirus genes, genes essential for the replication of coronavirus, West Nile virus genes, genes essential for the replication of West Nile virus, St. Louis encephalitis virus genes, genes essential for the replication of St. Louis encephalitis virus, tick-borne encephalitis virus genes, genes essential for the replication of tick-borne encephalitis virus, Murray Valley encephalitis virus genes, genes essential for the replication of Murray Valley encephalitis virus, dengue virus genes, genes essential for the replication of dengue virus, simian virus 40 genes, genes essential for the replication of simian virus 40, human T-cell lymphotropic virus genes, genes essential for the replication of human T-cell lymphotropic virus, Moloney murine leukemia virus genes, genes essential for the replication of Moloney murine leukemia virus, encephalomyocarditis virus genes, genes essential for the replication of encephalomyocarditis virus, measles virus genes, genes essential for the replication of measles virus, varicella-zoster virus genes, genes essential for the replication of varicella-zoster virus, adenovirus genes, genes essential for the replication of adenovirus, yellow fever virus genes, genes essential for the replication of yellow fever virus, poliovirus genes, genes essential for the replication of poliovirus, poxvirus genes, genes essential for the replication of poxvirus, malaria parasite genes, genes essential for the replication of malaria parasite genes, Mycobacterium ulcerans genes, genes essential for the replication of Mycobacterium ulcerans, Mycobacterium tuberculosis genes, genes essential for the replication of Mycobacterium tuberculosis, Mycobacterium leprae genes, genes essential for the replication of Mycobacterium leprae, Staphylococcus aureus genes, genes essential for the replication of Staphylococcus aureus, Streptococcus pneumoniae genes, genes essential for the replication of Streptococcus pneumoniae, Streptococcus pyogenes genes, genes essential for the replication of Streptococcus pyogenes, Chlamydia pneumoniae genes,Genes essential for Chlamydia pneumoniae replication, Mycoplasma pneumoniae genes, genes essential for Mycoplasma pneumoniae replication, integrin genes, selectin genes, complement system genes, chemokine genes, chemokine receptor genes, GCSF genes, Gro1 genes, Gro2 genes, Gro3 genes, PF4 genes, MIG genes, platelet-activating basic protein genes, MIP-1I genes, MIP-1J genes, RANTES genes, MCP-1 genes, MCP-2 genes, MCP-3 genes, CMBKR1 genes, CMBKR2 genes, CMBKR3 genes, CMBKR5v, AIF-1 genes, I-309 genes, genes for components of ion channels, genes for neurotransmitter receptors, genes for neurotransmitter ligands, amyloid family genes, presenilin genes, HD genes, DRPLA genes, SCA1 genes, SCA2 genes, MJD1 genes, CACNL1A4 genes, SCA7 genes, SCA8 genes, allelic genes found in loss of heterozygosity (LOH) cells, one allelic gene of polymorphic genes and combinations thereof, including but not limited to.

[0520] Loss of heterozygosity (LOH) can result in hemizygosity of sequences, such as genes, in the LOH region. This can result in significant genetic differences between normal and diseased state cells, such as cancer cells, and provides useful differences between normal and diseased state cells, such as cancer cells. This difference can be due to the fact that a gene or other sequence is heterozygous in diploid cells but hemizygous in cells with LOH. Regions of LOH often include loss of other sequences, such as genes that promote unwanted growth, such as tumor suppressor genes, and, for example, other genes that, in some cases, are essential for normal function, such as 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.

[0521] In certain embodiments, the present invention relates to oligonucleotides that modulate micro-RNAs.

[0522] CNS targeting In certain embodiments, the present invention provides oligonucleotides that target APP of early-onset familial Alzheimer's disease, ATXN2 of spinocerebellar ataxia type 2, and ALS and C9orf72 of amyotrophic lateral sclerosis and frontotemporal dementia.

[0523] In certain embodiments, the present invention provides oligonucleotides that target TARDBP of ALS, MAPT (Tau) of frontotemporal dementia, and HTT of Huntington's disease.

[0524] In certain embodiments, the present invention provides oligonucleotides that target SNCA of Parkinson's disease, FUS of ALS, ATXN3 of spinocerebellar ataxia type 3, ATXN1 of SCA1, genes of SCA7 and SCA8, ATN1 of DRPLA, MeCP2 of XLMR, PRNP of prion disease, autosomal recessive CNS disorder: Lafora disease, DMPK of DM1 (CNS and skeletal muscle), and TTR of hATTR (CNS, eye, and systemic).

[0525] Spinocerebellar ataxia is a genetic brain disorder. Dominant genetic forms of spinocerebellar ataxia such as SCA1-8 are devastating disorders without disease-modifying therapies. Examples of targets include SCA2, SCA3, and SCA1.

[0526] Further details regarding these CNS targeting receptors and related diseases can be found in PCT application PCT / US20 / 59399, entitled "Extrahepatic Delivery," filed on November 6, 2020, the content of which is hereby incorporated by reference in its entirety.

[0527] In certain embodiments, the present invention provides oligonucleotides that target genes of diseases including, but not limited to, age-related macular degeneration (AMD) (dry and wet forms), gyrate atrophy, dominant retinitis pigmentosa 4, Fuchs dystrophy, hATTR amyloidosis, genetic and sporadic glaucoma, and Stargardt disease.

[0528] In certain embodiments, the oligonucleotide targets VEGF in wet (or exudative) AMD.

[0529] In certain embodiments, the oligonucleotide targets C3 in dry (or nonexudative) AMD.

[0530] In certain embodiments, the oligonucleotide targets CFB in dry (or nonexudative) AMD.

[0531] In certain embodiments, the oligonucleotide targets MYOC in glaucoma.

[0532] In certain embodiments, the oligonucleotide targets ROCK2 in glaucoma.

[0533] In certain embodiments, the oligonucleotide targets ADRB2 in glaucoma.

[0534] In certain embodiments, the oligonucleotide targets CA2 in glaucoma.

[0535] In certain embodiments, the oligonucleotide targets CRYGC in cataract.

[0536] In certain embodiments, the oligonucleotide targets PPP3CB in dry eye syndrome.

[0537] Ligand In certain embodiments, the oligonucleotide is further modified by covalent attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties including, but not limited to, the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance of the attached compounds of the invention. Conjugate groups are routinely used in the chemical arts and are attached to the parent compound, such as an oligonucleotide, either directly or via an optional linking moiety or linker group. A list of preferred conjugate groups includes intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholanic acid moieties, folates, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, and dyes.

[0538] In certain embodiments, the oligonucleotide further comprises a targeting ligand that targets a receptor involved in delivery to a particular CNS tissue. These targeting ligands can also be conjugates combined with a lipophilic moiety to enable specific transcranial and systemic delivery.

[0539] Examples of targeting ligands that target receptor-mediated delivery to the CNS tissue are peptide ligands such as Angiopep-2, lipoprotein receptor-related protein (LRP) ligands, bEnd.3 cell-binding ligands; transferrin receptor (TfR) ligands (which can utilize the iron transport system in the brain and cargo transport to the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells, glial cells), glucose transporter proteins, and LDL receptor ligands.

[0540] In certain embodiments, the oligonucleotide further comprises a targeting ligand that targets a receptor involved in delivery to a particular eye tissue. These targeting ligands can also be conjugated in combination with a lipophilic moiety to enable specific eye delivery (e.g., intravitreal delivery) and systemic delivery. Examples of targeting ligands that target receptor-mediated delivery to eye tissue include lipophilic ligands such as all-trans retinol (which targets the retinoic acid receptor); RGD peptides (which target retinal pigment epithelial cells), such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO: 328) or cyclo(-Arg-Gly-Asp-D-Phe-Cys (SEQ ID NO: 329); LDL receptor ligands; and carbohydrate-based ligands (which target posterior eye endothelial cells).

[0541] Preferred conjugate groups applicable to the present invention include 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. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocolesterol (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); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., including 1996, 277, 923).

[0542] Generally, a wide variety of entities, such as ligands, can bind to the oligonucleotides described herein. Ligands can include naturally occurring molecules or recombinant or synthetic molecules. Examples of ligands are 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), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazene, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalators (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (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) cholenic acid, dimethoxytrityl or phenoxazine), peptide (e.g., alpha helix peptide, amphiphilic peptide, RGD peptide, cell-penetrating peptide, endosome destabilizing / fusion peptide), alkylating agent, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption promoter (e.g., naproxen, aspirin, vitamin, folic acid), synthetic ribonuclease (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu of tetraaza macrocycle, 3+ complex), dinitrophenyl, HRP, AP, antibody, hormone and hormone receptor, lectin, carbohydrate, polyvalent carbohydrate, vitamin (e.g., vitamin A, vitamin, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactor, lipopolysaccharide, MAP kinase activator, NF-κB activator, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, inadanocin, myoserpin, 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 permeating agent (e.g., helical cell permeating agent).

[0543] Peptides and peptidomimetic ligands include naturally occurring or modified peptides, such as those having D or L peptides; α, β or γ peptides; N-methyl peptides; azapeptides; one or more amides, i.e., peptide bonds, replaced with one or more urea, thiourea, carbamate or sulfonylurea bonds, i.e., peptides having peptide bonds; or cyclic peptides. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to that of a natural peptide. The peptide or peptidomimetic ligand can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length.

[0544] Examples of amphiphilic peptides include cecropin, ricin toxin, pardaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, serratotoxin, evoy (S. clava) peptide, nudaunagi intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, African clawed frog peptide, esculentin-1 and caerin.

[0545] As used herein, the term "endosome destabilizing ligand" refers to a molecule having endosome destabilizing properties. An endosome destabilizing ligand promotes the dissolution of the compositions or components of the present invention and / or the transport from cell compartments such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes or other vesicles within the cell to the cytoplasm of the cell. Some examples of endosome destabilizing ligands include, but are not limited to, imidazole, poly or oligoimidazole, linear or branched polyethyleneimine (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 having masked or unmasked cationic or anionic charges, dendrimers having masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptide mimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0546] Examples of endosome destabilization / fusion peptides include, but are not limited to, AALEALAEALEALAEALEALAEAAAAGGC(GALA) (SEQ ID NO: 330); AALAEALAEALAEALAEALAEALAAAAGGC(EALA) (SEQ ID NO: 331); ALEALAEALEALAEA (SEQ ID NO: 332); GLFEAIEGFIENGWEGMIWDYG(INF-7) (SEQ ID NO: 333); GLFGAIAGFIENGWEGMIDGWYG(InfHA-2) (SEQ ID NO: 334); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMIDGWYGC(diINF-7) (SEQ ID NO: 335); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC(diINF-3) (SEQ ID NO: 336); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC(GLF) (SEQ ID NO: 337); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC(GALA-INF3) (SEQ ID NO: 338); GLFEAIEGFIENGWEGnIDGKGLFEAIEGFIENGWEGnIDG(INF-5, Nis norleucine) (SEQ ID NO: 339); LFEALLELLESLWELLLEA(JTS-1) (SEQ ID NO: 340); GLFKALLKLLKSLWKLLLKA(ppTG1) (SEQ ID NO: 341); GLFRALLRLLRSLWRLLLRA(ppTG20) (SEQ ID NO: 342); WEAKLAKALAKALAKHLAKALAKALKACEA(KALA) (SEQ ID NO: 343); GLFFEAIAEFIEGGWEGLIEGC(HA) (SEQ ID NO: 344); GIGAVLKVLTTGLPALISWIKRKRQQ(Melittin) (SEQ ID NO: 345); H5WYG (SEQ ID NO: 346); and CHK6HC (SEQ ID NO: 347).

[0547] While not wishing to be bound by theory, fusogenic lipids fuse with and subsequently destabilize membranes. Fusogenic lipids typically have small headgroups and unsaturated acyl chains. Examples of fusogenic lipids include, but are not limited to, 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylcholine (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).

[0548] Synthetic polymers having endosomal destabilization activity applicable to the present invention are described in U.S. Patent Applications 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 hereby incorporated by reference in their entirety.

[0549] Examples of cell-penetrating peptides include, but are not limited to, RQIKIWFQNRRMKWKK (penetratin) (SEQ ID NO: 348); GRKKRRQRRRPPQC (Tat fragment 48-60) (SEQ ID NO: 349); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence-based peptide) (SEQ ID NO: 350); LLIILRRRIRKQAHAHSK (PVEC) (SEQ ID NO: 351); GWTLNSAGYLLKINLKALAALAKKIL (transportan) (SEQ ID NO: 352); KLALKLALKALKAALKLA (amphiphilic model peptide) (SEQ ID NO: 353); RRRRRRRRR (Arg9) (SEQ ID NO: 354); KFFKFFKFFK (bacterial cell wall-permeable peptide) (SEQ ID NO: 355); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37) (SEQ ID NO: 356); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO: 357); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO: 358); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO: 359); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID NO: 360); ILPWKWPWWPWRR-NH2 (indolicidin) (SEQ ID NO: 361); AAVALLPAVLLALLAP (RFGF) (SEQ ID NO: 362); AALLPVLLAAP (RFGF analog) (SEQ ID NO: 363); and RKCRIVVIRVCR (bactenecin) (SEQ ID NO: 364).

[0550] Examples of cationic groups include, for example, O-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, for example, O(CH2) nAmines (e.g., amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); amines (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); and NH(CH2CH2NH) n

[0551] As used herein, the term "targeting ligand" refers to a molecule that provides an increased affinity for a selected target, e.g., a cell, cell type, tissue, organ, body region or compartment, e.g., a cell tissue or organ compartment. Some examples of targeting ligands include, but are not limited to, antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.

[0552] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, polyvalent galactose, N-acetyl-D-galactosamine (GalNAc), polyvalent GalNAc, e.g., GalNAc2 and GalNAc3 (GalNAc and polyvalent GalNAc are collectively referred to herein as GalNAc conjugates); D-mannose, polyvalent mannose, polyvalent lactose, N-acetyl-glucosamine, glucose, polyvalent glucose, polyvalent fucose, glycosylated polyamino acids and lectins. The term polyvalent means that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other via glycosidic bonds or to a scaffold molecule.

[0553] Several folates and folate analogs applicable in the present invention as ligands are described in U.S. Patent Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the contents of which are hereby incorporated by reference in their entirety.

[0554] As used herein, the terms "PK modulating ligand" and "PK modulator" refer to molecules 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, PEG, biotin, and trans-thyretin binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligonucleotides containing some phosphorothioate sugar linkages are also known to bind to serum proteins, and thus short oligonucleotides, e.g., about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and oligonucleotides containing multiple phosphorothioate linkages in the backbone are also applicable as ligands (e.g., PK modulating ligands) in 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 nucleotide linkages in the PK modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. Additionally, aptamers that bind to serum components (e.g., serum proteins) are also applicable as PK modulating ligands in the present invention. Binding to serum components (e.g., 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.

[0555] When two or more ligands are present, the ligands can all have the same properties, all have different properties, or some ligands can have the same properties and others can have different properties. For example, the ligands can have targeting properties, endosomal destabilizing activity, or PK modulating properties. In a preferred embodiment, all the ligands have different properties.

[0556] The ligand or the tethered ligand can 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 one embodiment, the ligand can 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-terminal tether (i.e., having no associated ligand), e.g., monomer-linker-NH2, can be incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention). In a subsequent operation, i.e., after 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, e.g., a pentafluorophenyl ester or an aldehyde group, can be then attached to the precursor monomer by coupling of the electrophilic group of the ligand and the terminal nucleophilic group of the precursor monomer tether.

[0557] In other examples, a monomer having a chemical group suitable for participating in a click chemical reaction can be incorporated into, e.g., an azide or alkyne-terminal tether / linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the chain, a ligand having a complementary chemical group, e.g., an alkyne or an azide, can be attached to the precursor monomer by a copper-free click coupling of the alkyne and the azide.

[0558] In certain embodiments, the ligand can be conjugated to a nucleobase, sugar moiety, or internucleoside linkage of the oligonucleotide. Conjugation to a purine nucleobase or derivatives thereof can occur at any position including in - ring and exo - ring atoms. In certain embodiments, the 2 -, 6 -, 7 -, or 8 - position of the purine nucleobase is attached to the conjugate moiety. Conjugation to a pyrimidine nucleobase or derivatives thereof can also occur at any position. In certain embodiments, the 2 -, 5 -, and 6 - positions of the pyrimidine nucleobase can be substituted with the conjugate moiety. When the ligand conjugates to the nucleobase, the preferred positions are those that do not interfere with hybridization, i.e., do not interfere with the hydrogen - bonding interactions necessary for base pairing.

[0559] Conjugation to the sugar moiety of the nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached to the conjugate moiety can include the 2’, 3’, and 5’ carbon atoms. The 1’ - position can also be attached to the conjugate moiety, such as in the abasic residue. The internucleoside linkage can also carry the conjugate moiety. For phosphorus - containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom that is 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 of the amine or amide or to an adjacent carbon atom.

[0560] There are numerous methods for producing conjugates of oligonucleotides. Generally, the oligonucleotide is attached to the conjugate moiety by contact of a reactive group of the oligonucleotide (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) with a reactive group of the conjugate moiety. In certain embodiments, one reactive group is electrophilic and the other is nucleophilic.

[0561] For example, the electron-withdrawing group can be a carbonyl-containing functionality and the nucleophilic group can be an amine or a thiol. Methods for the conjugation of nucleic acids and related oligonucleotides with and without a linking group are well described in the literature such as, for example, 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.

[0562] The ligand can be attached to the oligonucleotide via a linker or a carrier monomer, e.g., a ligand carrier. The carrier includes (i) at least one "backbone attachment point", preferably two "backbone attachment points" and (ii) at least one "tethering attachment point". As used herein, a "backbone attachment point" is a functional group, e.g., a hydroxyl group or a bond that is generally available and suitable for incorporation into the backbone of the carrier monomer, e.g., a phosphate or modified phosphate of the oligonucleotide, e.g., a sulfur-containing backbone. A "tethering attachment point" (TAP) refers to an atom of the carrier monomer that attaches the selected moiety, e.g., a carbon atom or a heteroatom (different from the atom providing the backbone attachment point). The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is attached to the carrier monomer by an intervening tether. Thus, the carrier often contains a functional group, e.g., an amino group or provides a bond suitable for the incorporation or tethering of other chemical entities, e.g., constituent atoms of the ligand.

[0563] Representative U.S. patents teaching the manufacture of nucleic acid conjugates include, but are not limited to, U.S. Patent Nos. 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; 5,414,077; 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; the contents of which are hereby incorporated by reference in their entirety.

[0564] In certain embodiments, the oligonucleotide further comprises a targeting ligand that targets liver tissue. In certain embodiments, the targeting ligand is a carbohydrate-based ligand. In certain embodiments, the targeting ligand is a GalNAc conjugate.

[0565] In certain embodiments, the carbohydrate-based ligand is any of the ligands listed in Table 2, Table 2A, Table 3, Table 3A, Table 4, or Table 4A of WO2015 / 006740, which is hereby incorporated by reference in its entirety.

[0566] In certain embodiments, the linker, including a branched linker such as a divalent or trivalent branched linker for the attachment of these carbohydrate-based ligands, includes the linkers listed in Table 1 or Table 1A of WO2015 / 006740 and the spacers listed in Table 5, which is hereby incorporated by reference in its entirety.

[0567] In certain embodiments, the GalNAc-based conjugate is a GalNAc analog that contains an S or N atom or a -CH2- group in the glycosidic bond to convert a metabolically labile glycosidic bond, for example, the "O" in a glycosidic bond replaced with an S or N atom or a -CH2- group, to a metabolically stable glycosidic bond as shown in the scheme below.

Chemical formula

[0568] In certain embodiments, the GalNAc-based conjugate is a GalNAc analog having one of the following structures.

Table 37

Table 38

[0569] The GalNAc analogs listed in the above table can be produced using the methods described in WO2015 / 006740, which is hereby incorporated by reference in its entirety.

[0570] In certain embodiments, the GalNAc-based conjugate is a GalNAc analog having one of the following structures. [Chemical formula] [Chemical formula]

[0571] In certain embodiments, the oligonucleotide further comprises a ligand having the structure shown below: [Chemical formula] [wherein: L G is independently in each case a ligand, for example a carbohydrate, for example a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, polysaccharide; and Z’, Z’, Z’’ and Z’’’ are each independently in each case O or S. ].

[0572] In certain embodiments, the oligonucleotide comprises a ligand of formula (II), (III), (IV) or (V): [Chemical formula] [wherein: q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5Crepresents 0 to 20 independently in each case, where the repeating units may be the same or different; Q and Q’ are independently absent, -(P 7 -Q 7 -R 7 ) p -T 7 - or -T 7 -Q 7 -T 7’ -B-T 8’ -Q 8 -T 8 ; P 2A 、P 2B 、P 3A 、P 3B 、P 4A 、P 4B 、P 5A 、P 5B 、P 5C 、P 7 、T 2A 、T 2B 、T 3A 、T 3B 、T 4A 、T 4B 、T 4A 、T 5B 、T 5C 、T 7 、T 7’ 、T 8 and T 8’ are each independently absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O; B is -CH2-N(B L )-CH2-; B L is -T B -Q B -T B’ -R x ; ; Q 2A 、Q 2B 、Q 3A 、Q 3B 、Q 4A 、Q 4B 、Q 5A 、Q 5B 、Q 5C 、Q 7 、Q 8 and Q Bis independently absent, alkylene, or substituted alkylene in each case, where one or more methylenes may be interrupted or terminated by one or more of O, S, S(O), SO2, N(R N ), C(R’)=C(R’), C≡C, or C(O); T B and T B’ are each independently absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH2, CH2NH, or CH2O in each case; R x is lipophilic (e.g., 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) chenodeoxycholic acid, dimethoxytrityl, or phenoxazine), vitamin (e.g., folate, vitamin A, vitamin, biotin, pyridoxal), peptide, carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), endosome destabilizing component, steroid (e.g., uvaol, hecigenin, diosgenin), terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelinol derivative lithocholic acid), or cationic lipid; R 1 R 2 R 2A R 2B R 3A R 3B R 4A R 4B R 5A R 5B R 5C R 7 is independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=N-O,

Chemical formula

[0573] As described above, because the ligand can be conjugated to the oligonucleotide via a linker or a carrier and the linker or carrier can contain a branched linker, the oligonucleotide can contain multiple ligands via the same or different backbone attachment points to the carrier or via a branched linker. For example, the branch point of the branched linker can be a divalent, trivalent, tetravalent, pentavalent or hexavalent atom or a group presenting such multiple valences. In one embodiment, the branch point is -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C or -N(Q)C(O)O-C; where Q is independently H or alkyl optionally substituted in each case. In other embodiments, the branch point is glycerol or a glycerol derivative.

[0574] In certain embodiments, at least one ligand conjugated to the oligonucleotide is a transferrin receptor (TfR) ligand, such as a TfR1 ligand.

[0575] In certain embodiments, at least one ligand conjugated to the oligonucleotide is an integrin ligand (e.g., an integrin αvβ6 ligand or an integrin αVβ3 ligand).

[0576] In certain embodiments, the integrin ligand conjugated to the oligonucleotide is an integrin αVβ3 ligand. For example, the integrin ligand is

Chemical formula

Chemical formula

Chemical formula

[0577] In certain embodiments, the integrin ligand conjugated to the oligonucleotide is an integrin αVβ6 ligand.

[0578] In certain embodiments, the integrin ligand conjugated to the oligonucleotide is: R-G 1 -D-L-Xaa 1 -Xaa 2 -L-Xaa 3-Xaa 4 -L-R 1 (Formula VIII) (SEQ ID NO: 365) is an αvβ6 integrin ligand comprising: where: R is L-arginine; G 1 is L-glycine or N-methylglycine; D is L-aspartic acid (L-aspartate); L is L-leucine; Xaa 1 is L-alanine; Xaa 2 is L-α-amino-butyric acid (Abu); Xaa 3 is L-citrulline (Cit); Xaa 4 is α-amino-isobutyric acid (Aib); and R 1 is optional and, if present, comprises polyethylene glycol and / or a linking group.

[0579] In certain embodiments, in Formula VIII above, R 1 comprises polyethylene glycol having 2 to 20 ethylene oxide units. In certain embodiments, in Formula VIII above, the αvβ6 integrin ligand comprises the sequence Ac-RGDLAAbuLCitAibL (SEQ ID NO: 366).

[0580] In one embodiment, in the above formula VIII, the ανβ6 integrin ligand comprises an N-terminal cap. The N-terminal cap is CH3CO, CH3CH2CO, CH3(CH2)2CO, (CH3)2CHCO, CH3(CH2)3CO, (CH3)2CHCH2...

Claims

1. Compounds containing the structure of formula (I), or their salts or stereoisomers: [Cylindrical disulfide moiety] - [Phosphorus coupling group] (I) [In the formula, the [cyclic disulfide portion] is 【Chemistry 1】 It has the structure, and here: R １ is either 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 are each independently H, halo, CN or alkylene-CN, C(O)OR １３ or alkylene-C(O)OR １３ 、S(O)OR １３ or alkylene-S(O)OR １３ 、C(O)N(R')(R") or alkylene-C(O)N(R')(R"), OR １３ or alkylene-OR １３ 、N(R')(R") or alkylene-N(R')(R"), alkyl, C(R １４ )(R<7-digit tag unchanged>)(R １６ ) or alkylene-C(R １４ )(R １５ )(R １６ )、alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which may optionally be substituted with one or more R ｓｕｂ groups; R ３ and R ５ Each is independently H, halo, CN, or alkylene-CN, C(O)OR １３ or alkylene-C(O)OR １３ , S(O)OR １３ or alkylene-S(O)OR １３ , C(O)N(R')(R") or alkylene-C(O)N(R')(R"), 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 Often substituted with the base; or R ３ and R ５ It, together with the adjacent carbon atom and the two sulfur atoms, forms a second ring; R ２ and R ３ It may integrate with adjacent carbon atoms to form a further ring; R ４ and R ５ It may integrate with adjacent carbon atoms to form a further ring; R ６ and R ７ It may integrate with adjacent carbon atoms to form a further ring; R ８ and R ９ It may integrate with adjacent carbon atoms to form a further ring; R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １４ and R １５ Two or more of these may form one or more rings fused with a ring containing two sulfur atoms, together with adjacent carbon atoms; G is O, N(R'), S or C(R １４ )(R １５ ) and; n is an integer between 0 and 6; R １３ Each of these is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, each of which optionally contains one or more R ｓｕｂ Often substituted with the base; R １４ , R １５ and R １６ Each of these is independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, and N(R')(R”); R' and R'' are each independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, each of which optionally contains one or more R ｓｕｂ They are often substituted with groups; or R' and R'' form a ring together with the adjacent nitrogen atom, and R ｓｕｂ In each case, these are independently halo, haloalkyl, alkyl, alkalil, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, arylcarbonyl, acyloxy, cyano, or ureido.

2. In the [cyclic disulfide portion]: R １ O is; G is CH ２ And; n is 0 or 1; R ２ 、R ４ 、R ６ 、R ７ 、R ８ and R ９ are each independently H, halo, CN or C １ -C ６ -alkylene-CN, C(O)OR １３ or C １ -C ６ -alkylene-C(O)OR １３ 、S(O)OR １３ or C １ -C ６ -alkylene-S(O)OR １３ 、C(O)N(R')(R") or C １ -C ６ -alkylene-C(O)N(R')(R"), 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, heteroaryl, each of which may optionally be substituted with one or more R ｓｕｂ groups; R ３ and R ５ are each independently H, halo, CN or C １ -C ６ -alkylene-CN, C(O)OR １３ or C １ -C ６ -alkylene-C(O)OR １３ S(O)OR １３ or C １ -C ６ -alkylene-S(O)OR １３ C(O)N(R')(R") or C １ -C ６ -alkylene-C(O)N(R')(R"), 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, heteroaryl, each of which may optionally be substituted with one or more R ｓｕｂ groups; or R ３ and R ５ together with adjacent carbon atoms and two sulfur atoms form a second ring of 6 to 8 atoms; R ２ and R ３ These atoms may combine with adjacent carbon atoms to form a further ring of 3 to 7 atoms; R ４ and R ５ These atoms may combine with adjacent carbon atoms to form a further ring of 3 to 7 atoms; R ６ and R ７ These atoms may combine with adjacent carbon atoms to form a further ring of 3 to 7 atoms; R ８ and R ９ These atoms may combine with adjacent carbon atoms to form a further ring of 3 to 7 atoms; R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １４ and R １５ Two or more of these atoms can fuse with adjacent carbon atoms to form one or more rings of 5 to 7 atoms, condensed with a ring containing two sulfur atoms; R １３ In each case, H and C are independent. １ -C ６ It is alkyl, aryl, alkylcarbonyl or arylcarbonyl; and R' and R'' are each independently H or C １ -C ６ It is alkyl. The compound or a salt or stereoisomer thereof as described in claim 1.

3. [The cyclic disulfide portion] 【Chemistry 2】 [In the formula, n is between 1 and 4.] A compound according to claim 1, or a salt or stereoisomer thereof, having the structure described in claim 1.

4. R ２ and R ３ H and C are independent of each other. １ - ６ Alkyl, aryl, heteroaryl, CN, or CH ２ CN, OR １３ or CH ２ OR １３ , C(O)OR １３ or CH ２ C(O)OR １３ , S(O)OR １３ or CH ２ S(O)OR １３ , C(O)N(R')(R") or CH ２ C(O)N(R')(R") or C(R １４ )(R １５ )(R １６ ) or CH ２ C(R １４ )(R １５ )(R １６ ) and each of them may have one or more R ｓｕｂ Often substituted with the base; R １３ In each case, H and C are independent. １ - ６ They are alkyl, cycloalkyl, aryl, heteroaryl, or aralkyl; R １４ , R １５ and R １６ These are H, Haro, and C, respectively, and are independent of each other. １ - ６ It is alkyl, alkalic, aryl, or heteroaryl; and R' and R'' are H and C respectively, independently. １ - ６ It is alkyl, aryl, or heteroaryl. The compound or a salt or stereoisomer thereof as described in claim 3.

5. The [cyclic disulfide moiety] has the following structure: 【Transformation 3】 A compound according to claim 3 or a salt or stereoisomer thereof, having one of the above.

6. [Phosphorus coupling group] 【Chemistry 4】 [In the ceremony: X １ and Z １ Each of them independently represents H, OH, OM, OR １３ SH, SM, SR １３ , C(O)H, S(O)H, or alkyl (each preferably containing one or more R ｓｕｂ (may be substituted with the base), N(R')(R"), NSO ２ R', N=CN(R')(R"), B(R １３ ) ３ BH ３ － Se; or D-Q, where D is independently absent in each case, O, S, N(R'), alkylene, and each of them optionally contains one or more R ｓｕｂ It may be substituted with a group, and Q is independently a nucleoside or oligonucleotide in each case; X ２ and Z ２ Each of these is independent of N(R')(R"), OR １８ Alternatively, D-Q, where D is independently absent in each case, O, S, N, N(R'), alkylene, and each of them optionally contains one or more R ｓｕｂ It may be substituted with a group, and Q is independently a nucleoside or oligonucleotide in each case. Y １ is S, O, or N(R'); M is an organic or inorganic cation; and R １８ H or, if desired, one or more R ｓｕｂ It is an alkyl group substituted with a group. A compound according to claim 1, or a salt or stereoisomer thereof, having the structure described in claim 1.

7. The [phosphorus coupling group] has the following structure: 【Transformation 5】 【Transformation 6】 A compound according to claim 6 or a salt or stereoisomer thereof, having one of the above.

8. The compound according to claim 1, or a salt or stereoisomer thereof, wherein the compound has one of the following structures, and optionally the stereoisomer has a chiral purity of at least 70%. 【Transformation 7】 【Transformation 8】

9. One or more ligands, optionally via one or more linkers, R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ and R ９ A compound according to claim 1 or a salt or stereoisomer thereof, which is bonded to any of the following.

10. Structure of one or more equations (II): [Cylindrical disulfide moiety] - P(Y)(X) - ＊ (II) An oligonucleotide containing, where the [cyclic disulfide moiety] 【Chemistry 9】 or its salt or stereoisomer, Here: ＊ This represents binding to an oligonucleotide, Y is absent, N(R'), =O or =S, X is -OH, -SH, C(O)H, S(O)H, and one or more R as desired. ｓｕｂ alkyl groups, N(R')(R"), NSO2 substituted with a specific group. ２ R', N=CN(R')(R"), B(R １３ ) ３ BH ３ － or X', where X' is N(R')(R"), -OR １３ or -SR １３ And; R １ is O or S, and -P(Y)(X)- ＊ It is a bond to the P atom of the group; R ２ , R ４ , R ６ , R ７ , R ８ and R ９ Each of these independently is H, halo, CN, or alkylene-CN, C(O)OR １３ or alkylene-C(O)OR １３ , S(O)OR １３ or alkylene-S(O)OR １３ , C(O)N(R')(R") or alkylene-C(O)N(R')(R"), 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 Often substituted with the base; R ３ and R ５ Each of these independently is H, halo, CN, or alkylene-CN, C(O)OR １３ or alkylene-C(O)OR １３ , S(O)OR １３ or alkylene-S(O)OR １３ , C(O)N(R')(R") or alkylene-C(O)N(R')(R"), 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 Often substituted with the base; or R ３ and R ５ It combines with the adjacent carbon atom and two sulfur atoms to form a second ring; R ２ and R ３ It may integrate with adjacent carbon atoms to form a further ring; R ４ and R ５ It may integrate with adjacent carbon atoms to form a further ring; R ６ and R ７ It may integrate with adjacent carbon atoms to form a further ring; R ８ and R ９ It may integrate with adjacent carbon atoms to form a further ring; R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １４ and R １５ Two or more of these may become one or more rings fused with a ring containing two sulfur atoms, together with an adjacent carbon atom; G is O, N(R'), S or C(R １４ )(R １５ ) and; n is an integer from 0 to 6; R １３ In each case, independently, H is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, and each of them optionally contains one or more R ｓｕｂ Often substituted with the base; R １４ , R １５ and R １６ Each of these is independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, and N(R')(R”); R' and R'' are each independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, and each of them optionally contains one or more R ｓｕｂ Often substituted with a group; or R' and R'' unite with adjacent nitrogen atoms to form a ring, and R ｓｕｂ In each case, independently, these are halo, haloalkyl, alkyl, alkalil, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, 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 bound to the 5' end of the nucleoside or oligonucleotide. Oligonucleotides.

11. Formula: [Cylindrical disulfide moiety]-P(O)(SH)- ＊ [Cyclic disulfide moiety]-P(O)(OH)- ＊ [Cylindrical disulfide moiety]-P(O)(OR １３ )- ＊ [Cylindrical disulfide moiety]-P(S)(OR １３ )- ＊ [Cylindrical disulfide moiety] - P(S)(SH) - ＊ [Cylindrical disulfide moiety] - P(O)N(R')(R") - ＊ [Cylindrical disulfide moiety]-P(O)NSO ２ R'- ＊ [Cyclic disulfide moiety] - P(O)N = CN(R')(R")) - ＊ [Cylindrical disulfide moiety]-P(O)R １３ - ＊ The oligonucleotide according to claim 10, comprising a structure having the same structure or a salt thereof.

12. The following formula: 【Chemistry 10】 An oligonucleotide according to claim 11, comprising one of the structures.

13. The oligonucleotide comprises at least one [cyclic disulfide moiety] at the 5' end of the oligonucleotide, and the first nucleotide at the 5' end of the oligonucleotide is 【Chemistry 11】 [In the ceremony: R Ｓ is the [cyclic disulfide moiety]; X is -OH, -SH, C(O)H, S(O)H, and one or more R as desired. ｓｕｂ alkyl groups, N(R')(R"), NSO2 substituted with a specific group. ２ R', N=CN(R')(R"), B(R １３ ) ３ BH ３ － or X', where X' is N(R')(R"), -OR １３ or -SR １３ And, Y is S, O, or N(R'); Z is O, S, N(R') or CH ２ and The modified sugar is a sugar moiety containing one or more sugar modifications selected from the group consisting of 2' modification, LNA, isomer modification, 5' modification, unnatural cyclic modification, acyclic modification, and debasic modification. The oligonucleotide according to claim 11, having the structure of a salt or stereoisomer thereof.

14. The sugar modification is either 2' modification, LNA modification, isomer modification, 5' modification, or debase modification. Here, the first nucleotide at the 5' end of the oligonucleotide is 【Chemistry 12】 [In the ceremony: ＊ This represents binding to a nucleotide bond modified as desired; B is a modified nucleic acid base or H as desired; R Ｓ is the [cyclic disulfide moiety]; and R １ H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH ２ -Allyl, fluoro, O-N-methylacetamide (O-NMA), O-N-alkylacetamide, O-dimethoxypropyl, O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP), or ara-F; or R １ It forms a bridge with the 4' carbon; R ２ is H, alkyl, or aryl; X is -OH, -SH, C(O)H, S(O)H, and one or more R as desired. ｓｕｂ alkyl groups, N(R')(R"), NSO2 substituted with a specific group. ２ R', N=CN(R')(R"), B(R １３ ) ３ BH ３ － or X', where X' is N(R')(R"), -OR １３ or -SR １３ And, Y is S, O, or N(R'); Z is O, S, N(R') or CH ２ and Q is O, S, CH ２ Or it is N(R'). The oligonucleotide according to claim 13, having the structure of a salt or stereoisomer thereof.

15. The following structures are formed when the first nucleotide at the 5' end of an oligonucleotide is selected from groups (i) to (v): (i) 【Chemistry 13】 (ii) 【Chemistry 14】 (iii) 【Chemistry 15】 (iv) 【Chemistry 16】 and (v) 【Chemistry 17】 The oligonucleotide according to claim 14, having one of the following.

16. [Modified sugar] has the following structure: (a) [Chemistry 18] [In the ceremony: ＊ This represents binding to a nucleotide bond modified as desired; and B is a modified nucleic acid base or H as desired. Includes and / or unnatural cyclic modifications having one of the following: (b) 【Chemistry 19】 [In the ceremony: * represents binding to a nucleotide bond modified as desired; and B is a modified nucleic acid base or H as desired. Includes acyclic modifications having one of the following: The oligonucleotide according to claim 13.

17. The oligonucleotide comprises at least one [cyclic disulfide moiety] at the 5' end of the oligonucleotide, wherein the first nucleotide at the 5' end of the oligonucleotide is structured as follows: 【Chemistry 20】 or has a salt or stereoisomer thereof, where ＊ This represents binding to a nucleotide bond modified as desired; Base is a nucleic acid base that has been modified as desired; R Ｓ is the [cyclic disulfide moiety]; and R is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH ２ - Allyl, fluoro, O-N-methylacetamide (O-NMA), O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP), or ara-F The oligonucleotide according to claim 10.

18. A pharmaceutical composition comprising the oligonucleotide described in claim 10 and a pharmaceutically acceptable additive.

19. The pharmaceutical composition according to claim 18 for reducing or inhibiting the expression of a target gene in a subject.

20. A precursor compound containing the structure of formula (I), or a salt or stereoisomer thereof: [Cyclic disulfide moiety]–[Phosphorus coupling group](I) Here, the [cyclic disulfide portion] 【Chemistry 21】 [In the ceremony: R １ It is either O or S and is bonded to the P atom of the [phosphorus coupling group]; R ２ (CH ２ ) ｓ -W, (CH ２ ) ｓ -O-(CH ２ ) ｓ -W, (CH ２ ) ｓ -(CH ２ CH ２ O) ｔ -(CH ２ ) ｓ -W or (CH ２ ) ｓ O(CH ２ CH ２ O) ｔ -(CH ２ ) ｓ -W is; s is an integer between 0 and 22, t is an integer between 1 and 20; W is a reactive group; R ４ and R ５ Each of these independently is H, halo, CN, or alkylene-CN, C(O)OR １３ or alkylene-C(O)OR １３ , S(O)OR １３ or alkylene-S(O)OR １３ , C(O)N(R')(R") or alkylene-C(O)N(R')(R"), 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 Often substituted with the base; or R ４ and R ５ This forms 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 In each case, independently, H is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, and each of them optionally contains one or more R ｓｕｂ Often substituted with the base; R １４ , R １５ and R １６ Each of these is independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, and N(R')(R”); R' and R'' are each independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, and each of them optionally contains one or more R ｓｕｂ They are often substituted with a group; or R' and R'' form a ring with the adjacent nitrogen atom; and R ｓｕｂ In each case, these are independently halo, haloalkyl, alkyl, alkalil, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, arylcarbonyl, acyloxy, cyano, or ureido. A precursor compound having the structure thereof, or a salt or stereoisomer thereof.

21. [The cyclic disulfide portion] 【Chemistry 22】 [In the formula, R ４ and R ５ H and C are independent of each other. １ - ６ Alkyl or phenyl; or R ４ and R ５ It combines with an adjacent carbon atom to form a second ring of 3 to 7 atoms. A precursor compound or a salt or stereoisomer thereof according to claim 20, having the structure described above.

22. R ２ In this case, W is NHTFA, N ３ , C≡CH, C(O)OR １３ or OC(O)R １３ And here, R １３ C １ -C ３ The precursor compound or a salt or stereoisomer thereof according to claim 20, wherein the precursor compound is alkyl.

23. [Phosphorus coupling group] 【Chemistry 23】 [In the ceremony: X １ and Z １ Each of them independently represents H, OH, OM, OR １３ SH, SM, SR １３ , C(O)H, S(O)H, or alkyl (each preferably containing one or more R ｓｕｂ (may be substituted with the base), N(R')(R"), NSO ２ R', B(R １３ ) ３ BH ３ － Se; or D-Q, where D is independently absent in each case, O, S, N(R'), alkylene, and each of them optionally contains one or more R ｓｕｂ It may be substituted with a group, and Q is independently a nucleoside or oligonucleotide in each case; X ２ and Z ２ Each of these is independent of N(R')(R"), OR １８ Alternatively, D-Q, where D is independently absent in each case, O, S, N, N(R'), alkylene, and each of them optionally contains one or more R ｓｕｂ It may be substituted with a group, and Q is independently a nucleoside or oligonucleotide in each case. Y １ is S, O, or N(R'); M is an organic or inorganic cation; and R １８ H or, if desired, one or more R ｓｕｂ It is an alkyl group substituted with a group. A precursor compound or a salt or stereoisomer thereof according to claim 20, having the structure described above.

24. Formula (II): [Cylindrical disulfide moiety] - P(Y)(X) - ＊ (II) or an oligonucleotide comprising one or more structures of a salt or stereoisomer thereof, wherein the [cyclic disulfide moiety] 【Chemistry 24】 [In the ceremony: ＊ This represents binding to an oligonucleotide, Y is absent, N(R'), =O or =S, X is -OH, -SH, C(O)H, S(O)H, and one or more R as desired. ｓｕｂ alkyl groups, N(R')(R"), NSO2 substituted with a specific group. ２ R', N=CN(R')(R"), B(R １３ ) ３ BH ３ － or X', where X' is N(R')(R"), -OR １３ or -SR １３ And; R １ is O or S, and -P(Y)(X)- ＊ It is a bond to the P atom of the group; R ２ (CH ２ ) ｓ -W, (CH ２ ) ｓ -O-(CH ２ ) ｓ -W, (CH ２ ) ｓ -(CH ２ CH ２ O) ｔ -(CH ２ ) ｓ -W or (CH ２ ) ｓ O(CH ２ CH ２ O) ｔ -(CH ２ ) ｓ -W is; s is an integer between 0 and 22, t is an integer between 1 and 20; W is a reactive group; R ４ and R ５ Each of these independently is H, halo, CN, or alkylene-CN, C(O)OR １３ or alkylene-C(O)OR １３ , S(O)OR １３ or alkylene-S(O)OR １３ , C(O)N(R')(R") or alkylene-C(O)N(R')(R"), 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 Often substituted with the base; or R ４ and R ５ This forms 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 In each case, independently, H is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, aralkyl, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, and each of them optionally contains one or more R ｓｕｂ Often substituted with the base; R １４ , R １５ and R １６ Each of these is independently H, halo, haloalkyl, alkyl, alkaryl, aryl, heteroaryl, aralkyl, hydroxy, alkyloxy, aryloxy, and N(R')(R”); R' and R'' are each independently H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, hydroxy, alkyloxy, ω-aminoalkyl, ω-hydroxyalkyl, ω-hydroxyalkenyl, or ω-hydroxyalkynyl, and each of them optionally contains one or more R ｓｕｂ They are often substituted with a group; or R' and R'' form a ring together with the adjacent nitrogen atom, and R ｓｕｂ In each case, independently, these are halo, haloalkyl, alkyl, alkalil, aryl, aralkyl, hydroxy, alkyloxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, arylcarbonyl, acyloxy, cyano, or ureido; Here, at least one [cyclic disulfide moiety] is attached to the 5' end of the nucleoside or oligonucleotide. Oligonucleotides having a specific structure.

25. [The cyclic disulfide portion] 【Chemistry 25】 [In the formula, R ４ and R ５ H and C are independent of each other. １ - ６ Alkyl or phenyl; or R ４ and R ５ It combines with an adjacent carbon atom to form a second ring of 3 to 7 atoms. The oligonucleotide according to claim 24, having the structure.

26. R ２ In this case, W is NHTFA, N ３ , C≡CH, C(O)OR １３ OC(O)R １３ And here, R １３ C １ -C ３ The oligonucleotide according to claim 24, wherein it is alkyl.

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