Single-chain loop oligonucleotide

Abstract

One aspect of the present invention relates to a single-stranded oligonucleotide having a sequence represented by formula (II) or (III): (5'-Z 11 -3')-L-Q S -(5'-Z 12 -3')(II), (3'-Z 11 -5')-L-Q S -(3'-Z 12 -5')(III). In formula (II) or (III), Z 11 is a first oligonucleotide comprising 15 to 100 optionally modified nucleotides that is substantially complementary to the target gene; Z 12 is a second oligonucleotide comprising 10 to 100 optionally modified nucleotides that is substantially complementary to Z 11 ; Z 11 and Z 12 can form an intrastrand double-stranded region containing 7 or more consecutive base pairs; Q S represents 0 to 12 optionally modified nucleotides; L is an arbitrary linking group; at least one nucleotide of formula (II) is a modified nucleotide; at least one nucleotide of formula (III) is a modified nucleotide; at least one nucleotide at the 3'-end of Z 11 in formula (II), or at least one nucleotide at the 5'-end of Z 11 in formula (III), in either case together with L and Q S forms a loop region connecting Z 11 and Z 12 .

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 466,214, filed on 12 May 2023, which is incorporated herein by reference in its entirety.

[0002] Array List This application includes an array list submitted in XML format, which is incorporated herein by reference in its entirety. The XML copy prepared on 7 May 2024 is named 29520_1515-PCT_ALN-509_SL.xml and has a size of 1,690,493 bytes.

[0003] This invention generally relates to the field of RNA interference technology using single-stranded loop oligonucleotides. [Background technology]

[0004] Chemical modifications of nucleobases, ribose sugars, and phosphate backbones are used in double-stranded RNAi agents to improve the drug-like properties of these therapeutic oligonucleotides and confer favorable pharmacological properties to GalNAc oligonucleotide conjugates in preclinical and clinical development.

[0005] Various siRNA designs have been developed to achieve better stability and potency. Current research has addressed stability and duration-related challenges by incorporating chemical modifications, but has overlooked process-related challenges in the synthesis of double-stranded siRNA. Sense and antisense strands are typically synthesized separately, undergo cumbersome multi-step purification as single strands, and then annealed to double strands, which then undergo further purification and quality control. This process is complex, time-consuming, expensive, and raises environmental sustainability concerns.

[0006] However, there is still a need for improved designs of RNAi agents that simplify the manufacturing and purification processes while simultaneously preserving or improving the effectiveness of the RNAi agent. SUMMARY OF THE INVENTION

[0007] One aspect of the invention is a compound of formula (I): (5’-Z 1 -³’)-Q 1 -L-Q 2 -(5’-Z 2 -³’) (I) [wherein: Z 1 is a first oligonucleotide comprising 10 to 100 appropriately modified nucleotides (e.g., 15 to 100) that are substantially complementary to the target gene; Z 2 is a second oligonucleotide comprising 10 to 100 appropriately modified nucleotides (e.g., 15 to 100) that are substantially complementary to Z 1 ; Z 1 and Z 2 can form an intrastrand duplex region containing 3 or more consecutive base pairs; L is a linking group; Q 1 and Q 2 each independently represent 0 to 12 appropriately modified nucleotides] A single-stranded oligonucleotide having a sequence represented by can inhibit the expression of a target gene, where at least one nucleotide of formula (I) is a modified nucleotide, relates to a single-stranded oligonucleotide.

[0008] The first oligonucleotide Z 1 and the second oligonucleotide Z 2 can each independently contain 15 to 100 appropriately modified nucleotides. For example, Z 1 and Z 2Each may independently contain 15-40, 15-25, or 19-23 appropriately modified nucleotides. In some embodiments, the first oligonucleotide Z 1 and the second oligonucleotide Z 2 Each of these may independently contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotide lengths. 1 and Z 2 Each of these can independently have a length of approximately 10 to 50 nucleotides, approximately 10 to 40 nucleotides, approximately 15 to 40 nucleotides, approximately 10 to 35 nucleotides, approximately 10 to 30 nucleotides, approximately 10 to 25 nucleotides, approximately 10 to 20 nucleotides, approximately 15 to 50 nucleotides, approximately 15 to 40 nucleotides, approximately 15 to 35 nucleotides, approximately 15 to 30 nucleotides, approximately 15 to 25 nucleotides, approximately 15 to 20 nucleotides, approximately 19 to 23 nucleotides, approximately 19 to 21 nucleotides, or approximately 18 to 20 nucleotides. 1 and the second oligonucleotide Z 2 Each nucleotide can be modified independently and as appropriate. In some embodiments, Z 1 and Z 2 Each contains the same number of appropriately modified nucleotides.

[0009] Q 1 and Q 2 Each of these can independently contain 0 to 12 appropriately modified nucleotides. For example, Q 1 and Q 2 Each of these may independently contain appropriately modified nucleotides of 0-10, 0-6, 0-4, 0-3, 0-2, 1-6, 1-4, 1-3, or 2-3. In some embodiments, Q 1 and Q 2 These are all 0. In some embodiments, Q 1 and Q2 One of them is 0. In some embodiments, Q 1 and Q 2 It has the same number of appropriately modified nucleotides.

[0010] In some embodiments, a single-stranded oligonucleotide can be cleaved at a linking group L. First oligonucleotide Z 1 It is cleaved into an antisense strand that is substantially complementary to the target gene (e.g., target mRNA or DNA), and a second oligonucleotide Z 2 is, Z 1 It can be cut into a sense chain that is substantially complementary to it.

[0011] First oligonucleotide Z 1 and the second oligonucleotide Z 2 This can form intramolecular double-stranded regions containing three or more consecutive base pairs (e.g., double-stranded regions of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs). For example, the double-stranded regions may contain 10-25, 15-25, 19-23, 19, 20, 21, 22, or 23 base pairs. 1 and Z 2 The intrachain double-strand region formed by this may contain all consecutive base pairs or three or fewer mismatched base pairs (e.g., 0, 1, 2, or 3).

[0012] In some embodiments, the single-stranded oligonucleotide includes at least one chemical modification. In some embodiments, the first oligonucleotide Z 1 and the second oligonucleotide Z 2 Each of these includes at least one chemical modification. In some embodiments, Z 2 All of the nucleotides are modified nucleotides. In some embodiments, Z 1 All of the nucleotides are modified nucleotides. In some embodiments, all nucleotides of a single-stranded oligonucleotide are modified.

[0013] Chemical modifications to nucleotides may include nucleoside linkage modifications, nucleobase modifications, sugar modifications, or combinations thereof.

[0014] In certain embodiments, the chemical modification is selected from the group consisting of LNA, ENA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-alkyl (e.g., 2'-O-methyl), 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modification (e.g., 2'-modified L-nucleoside, e.g., 2'-deoxy-L-nucleoside), BNA debasic sugars, debasic cyclic and open-chain alkyls, and combinations thereof.

[0015] In certain embodiments, the chemical modification is selected from the group consisting of at least one modified nucleotide, and includes deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides (e.g., LNA), unlocked nucleotides (e.g., UNA), structurally restricted nucleotides, restricted ethyl nucleotides, debasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramides, non-natural bases including nucleotides, and tetrahydro These are pyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing 5'-phosphate or a 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleotides containing glycol nucleic acid (GNA), nucleotides containing glycol nucleic acid (GNA) S isomers (S-GNA), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-5'-linked nucleotides ("3'-RNA"), or terminal nucleotides linked to cholesteryl derivatives or dodecanoate bisdecylamide groups.

[0016] In certain embodiments, the chemical modification is a 2'-modification selected from the group consisting of 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, 2'-fluoro, and combinations thereof.

[0017] In some embodiments, Z is approximately 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30%. 1 It is modified. In some embodiments, Z 2 Approximately 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of Z 2 The nucleotides are modified. In some embodiments, about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of all nucleotides in the single-stranded oligonucleotide are modified. For example, if 50% of all nucleotides are modified, then 50% of all nucleotides present in the single-stranded oligonucleotide contain at least one modification described herein.

[0018] In one embodiment, at least 50% of the nucleotides of a single-stranded oligonucleotide are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, or 2'-fluoro.

[0019] In some embodiments, one or more of the five internucleotide links within the six 3'-terminal nucleotides are modified internucleotide links. In some embodiments, one or more of the five internucleotide links within the six 5'-terminal nucleotides are modified internucleotide links.

[0020] In some embodiments, Z 2 One or more of the five internucleotide links within the six 5'-terminal nucleotides are modified internucleotide links. In some embodiments, Z 1 One or more of the five internucleotide junctions within the six 5'-terminal nucleotides are modified internucleotide junctions.

[0021] In some embodiments, the single-stranded oligonucleotide is Z 1 The 3'-terminal nucleotide and Q 1 The first nucleotide further comprises one or more modified nucleotide links. In some embodiments, the single-stranded oligonucleotide is Q 1 The nucleotides further include one or more modified internucleotide links.

[0022] In some embodiments, the single-stranded oligonucleotide further comprises a phosphate group or a phosphate mimetic at the 5' end of the nucleotide sequence (e.g., Z 1 and / or Z 2 ). In some embodiments, the single-stranded oligonucleotide contains a phosphate mimetic at the 5' end of the nucleotide sequence (e.g., Z 1 and / or Z 2 In one embodiment, at least one phosphate mimetic is Z 1 It is located at the 5' end. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP). In one embodiment, the phosphate mimetic is 5'-cyclopropylphosphonate. In one embodiment, the phosphate mimetic is 5'-vinylphosphate.

[0023] In some embodiments, the 5' or 3' terminal nucleotide of the single-stranded oligonucleotide of formula (I) includes a 2'-5'-linked nucleotide modification; or the 5' or 3' terminal nucleotide is conjugated to a debasalized nucleotide, an inverted nucleotide, or an inverted debasalized nucleotide (e.g., ribonucleotide) via a phosphodiester, phosphorothioate, or phosphodithioate linkage, as appropriate.

[0024] In some embodiments, the 5' or 3' terminal nucleotide of the single-stranded oligonucleotide of formula (I) is modified and includes a linking moiety containing mono-, di-, tri-, tetra-, penta-, or polyprolinol, or mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinol.

[0025] In some embodiments, the single-stranded oligonucleotide further comprises at least one terminal, chiral modification (e.g., terminal, chiral phosphorus atom).

[0026] Site-specific, chiral modifications to nucleotide linkages can be present at the 5' end, 3' end, or both ends of a nucleotide sequence. These are referred to herein as “terminal, chiral” modifications. Terminal modifications can be located at the 3' or 5' terminal position of the terminal region, for example, at the terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide sequence. Each chiral pure phosphorus atom can be in either an Rp configuration, an Sp configuration, or a combination thereof. Further details regarding chiral modifications and chiral-modified RNA agents can be found in WO2019 / 126651A1, which is incorporated herein by reference in its entirety.

[0027] In some embodiments, the single-stranded oligonucleotide comprises at least two sequential phosphorothioate or methylphosphonate nucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least two blocks of two sequential phosphorothioate or methylphosphonate nucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least three blocks of two sequential phosphorothioate or methylphosphonate nucleotide linkage modifications.

[0028] In some embodiments, a single-stranded oligonucleotide has at least two phosphorothioate nucleotide linkages in the first five nucleotides of the nucleotide sequence (counting from the 5' end) (e.g., Z 1 and / or Z 2 ).

[0029] In some embodiments, single-stranded oligonucleotides (e.g., Z 1 and / or Z 2The nucleotide sequence of ) contains two blocks of one, two, or three phosphorothioate nucleotide links separated by phosphate nucleotide links of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0030] In one embodiment, a single-stranded oligonucleotide (e.g., Z 1 and / or Z 2 The nucleotide sequence of (e.g., Z) contains at least two consecutive phosphorothioate nucleotide linkage modifications within the 18-23 position of the nucleotide sequence, counting from the 5' end of the nucleotide sequence. In one embodiment, a single-stranded oligonucleotide (e.g., Z) 1 and / or Z 2 The nucleotide sequence of ) contains at least two consecutive phosphorothioate nucleotide ligation modifications within positions 1 to 5 of the nucleotide sequence, counted from the 5' end of the nucleotide sequence.

[0031] In some embodiments, the Z of a single-stranded oligonucleotide 1 and Z 2 Each of these includes at least two consecutive phosphorothioate nucleotide ligation modifications. In one embodiment, the Z of a single-strand oligonucleotide 1 and Z 2 Each of these includes at least two consecutive phosphorothioate internucleotide ligation modifications within positions 18–23 of the nucleotide sequence, and at least two consecutive phosphorothioate internucleotide ligation modifications within positions 1–5 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence.

[0032] In all of the embodiments described above, the target gene may be mRNA, pre-mRNA, microRNA, pre-miRNA, long non-coding RNA (lncRNA), or DNA.

[0033] In all of the embodiments described above, the single-stranded oligonucleotide may be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, a microRNA mimetic, a supermir, an aptamer, a U1 adapter, a triple-stranded oligonucleotide, an RNA activator, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a heterodouble-stranded oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide.

[0034] In some embodiments, single-stranded oligonucleotides (e.g., Z 1 and / or Z 2 Each of the at least one, two, three, four, or five terminal phosphorus-containing links at the 5' or 3' end of ) is not a phosphorothioate linkage. In one embodiment, a single-stranded oligonucleotide (e.g., Z 1 and / or Z 2 Each of the at least one, two, three, four, or five terminal phosphorus-containing linkages at the 5' or 3' end of ) is independently either a natural phosphate group or a phosphodiester linkage, or a nitrogen-modified phosphorus-containing linkage (PN-linkage).

[0035] In some embodiments, the PN linkage is expressed as -N(R)P(=X)(OH)O- or -OP(=X)(OH)N(R)-, -OP(NR)(=X)O-, N(SO2R)P(=X)(OH)O- or -OP(=X)(OH)N(SO2R)-, or -OP(NSO2R)(=X)O- [wherein X is O or S; R may be an appropriately substituted alkyl, aryl, heteroaryl or heterocyclyl; or NR may be an appropriately substituted cyclic guanidine moiety, an appropriately substituted triazolyl group or Tmg group ( [ka] ) may have [possibly be].

[0036] In some embodiments, the PN linkage includes a cyclic guaninidine moiety that is appropriately substituted. For example, the PN linkage is [ka] The structure may be [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the PN linkage is stereochemically controlled.

[0037] In some embodiments, the PN linkage includes a triazole moiety (e.g., a suitably substituted triazole group). For example, the PN linkage is [ka] The structure may be [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the PN linkage is stereochemically controlled.

[0038] In some embodiments, the PN linkage includes an alkyne moiety (e.g., an appropriately substituted alkynyl group). For example, the PN linkage is [ka] The structure may be [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the PN linkage is stereochemically controlled.

[0039] In some embodiments, the PN linkage is a Tmg group ( [ka] ) includes. For example, PN consolidation is [ka] The structure may be [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the PN linkage is stereochemically controlled.

[0040] Further preferred PN consolidations may include those described in WO2019 / 032612 and WO2021 / 030778, which are incorporated herein by reference in their entirety.

[0041] In some embodiments, L in formula (I) is a cleavage linker. In some embodiments, the cleavage linker is cleavable in homogenates, tritosomes, cytosols, or endosomes of any type of cell. For example, the cleavage linker may be cleavable in liver homogenates, liver tritosomes, liver lysosomes, liver cytosols, liver endosomes, brain homogenates, brain tritosomes, brain lysosomes, brain cytosols, or brain endosomes. In certain embodiments, the cleavage linker is a redox cleavage linker (e.g., a reductive cleavage linker; e.g., a disulfide group), an acid cleavage linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavage linker (e.g., an ester group), a phosphatase cleavage linker (e.g., an ester group), a peptidase cleavage linker (e.g., an ester group), or an endosome cleavage linker (or a protease cleavage linker, e.g., a carbohydrate linker).

[0042] In some embodiments, the cleavage linker (tether) is an endosomal cleavage linker or a protease cleavage linker, such as a carbohydrate linker, which is cleaved at least 1.25 times 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).

[0043] In some embodiments, L in formula (I) is equal to formula #-(N) n - **It contains a connecting part represented by . In this formula, # is Q 1 It is a coupling to, ** Q 2 It is a bond to; n is 3 to 12; and each N is independently a linking part. For example, each N can independently be a linked monomer with a chain length of 3 or more atoms.

[0044] In this specification, the term "chain length" refers to the number of atoms in the shortest linear chain formed by linked monomers. For example, [ka] In PEG / PEO, which has the structure shown, the chain length of the linked monomer is 3 (triethylene glycol). Another example is the peptide linked monomer, [ka] The chain length of a linked monomer having the formula is 3. In one embodiment, [ka] The chain length of a linked monomer having the formula is 6. In one embodiment, [ka] The chain length of a linked monomer having the formula is 7. In one embodiment, [ka] The chain length of a linked monomer having the formula is 13.

[0045] In some embodiments, one or more linking portions (N) in L of formula (I) may be appropriately modified nucleotides.

[0046] In some embodiments, one or more linkages in L of formula (I) may be independently selected from the group consisting of 2'-deoxynucleotides (dN), 2'-deoxy-2'-fluoronucleotides (fN), ribonucleotides (rN), 2'-O-methylnucleotides (mN), and 2'-ara nucleotides (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide). Ara nucleotides have opposite stereochemical characteristics at the 2' carbon atom compared to ribonucleotides.

[0047] In certain embodiments, one or more linking portions (N) in L of formula (I) are phosphodiesters, phosphotriesters (which may contain a linked phosphorus atom in either the Rp or Sp configuration), hydrogen phosphonates, alkyl or aryl phosphonates, phosphoramidates (which may contain a linked phosphorus atom in either the Rp or Sp configuration), phosphorothioates (which may contain a linked phosphorus atom in either the Rp or Sp configuration), methylenemethyliminos, nitrogen-modified phosphorus-containing linkages (PN linkages) (which may contain a linked phosphorus atom in either the Rp or Sp configuration), thiodiesters, thionocarbamates, N,N'-dimethylhydrazines, phosphoroselenates, boranophosphates. It may contain modified nucleotide linkages selected from the group consisting of phosphate, boranophosphate esters, amides, hydroxylaminos, siloxanes, dialkylsiloxanes, carboxamides, carbonates, carboxymethyls, carbamates, carboxylate esters, thioethers, ethylene oxide linkers, sulfides, sulfonates, sulfonamides, sulfonate esters, thioformacetals, formacetals, oximes, methyleneiminos, methylenecarbonylaminos, methylenehydrazos, methylenedimethylhydrazos, methyleneoxymethyliminos, ethers, thioacetamides, and combinations thereof.

[0048] In certain embodiments, one or more linking portions (N) in L of formula (I) may contain a portion selected from the group consisting of: aliphatic saturated or unsaturated alkyl chains; phosphorus-containing links including phosphoric acid, phosphonates, phosphoramidates (which may contain linked phosphorus atoms in either the Rp or Sp configuration), phosphodiesters, phosphotryesters (which may contain linked phosphorus atoms in either the Rp or Sp configuration), phosphorothioates (which may contain linked phosphorus atoms in either the Rp or Sp configuration), and nitrogen-modified phosphorus-containing links (PN links) (which may contain linked phosphorus atoms in either the Rp or Sp configuration); (poly)ethylene glycol chains including diethylene glycol, triethylene glycol, tetra, penta, hexa, hepta, octa, nona, or decaethylene glycol; glycerol or glycerol esters; aminoalkyl ethers; and combinations thereof.

[0049] In some embodiments, one or more linking portions (N) in L of formula (I) may contain portions selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, as well as combinations thereof.

[0050] In some embodiments, one or more connecting portions (N) in L of formula (I) are independently [ka] A selection can be made from the group consisting of the following.

[0051] In some embodiments, one or more connecting portions (N) in L of formula (I) are independently [ka] [In formula: Base is a nucleobase that has been modified as appropriate. R D C4~ 30 Alkyl, C4~ 30Alkenyl, or C4~ 30 [It is Alkinnil] A selection can be made from the group consisting of the following.

[0052] In some embodiments, one or more linkage portions (N) in L of formula (I) include mono, di, tri, tetra, penta, or polyprolinol appropriately conjugated with a ligand; mono, di, tri, tetra, penta, or polyhydroxyprolinol appropriately conjugated with a ligand; appropriately modified nucleotides; or combinations thereof.

[0053] In some embodiments, L in formula (I) comprises one or more mono, di, tri, tetra, penta, or polyprolinols, optionally conjugated with a ligand; and one or more optionally modified nucleotides.

[0054] In some embodiments, L in formula (I) comprises one or more mono, di, tri, tetra, penta, or polyhydroxyprolinols, optionally conjugated with a ligand; and one or more optionally modified nucleotides.

[0055] In some embodiments, one or more connecting portions (N) in L of formula (I) are [Table 1-1] [Table 1-2] Includes a portion selected from the group consisting of

[0056] In some embodiments, L in formula (I) is equal to formula #-(N) n - ** It contains a connecting part represented by . In this formula, # is Q 1 It is a coupling to, ** Q 2is a bond to; n is from 3 to 12; each N is, independently, an appropriately modified nucleotide, Y34, Y16, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, or Q368. In some embodiments, n is from 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5. In one embodiment, n is 5.

[0057] In some embodiments, L of formula (I) contains 3 to 5 2'-deoxynucleotides, a triplet of 2'-deoxy-2'-fluoronucleotides, a triplet of ribonucleotides, a triplet of 2'-O-methylnucleotides, or a triplet of Q304.

[0058] In some embodiments, L of formula (I) is as follows: #-mN-mN-mN-mN-mN- ** , [[ID=...]] (The remaining content can be translated in a similar way following the above rules for each line.)​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​rN represents ribonucleotide, mN represents 2'-O-methylnucleotide. It contains one of the following.

[0059] In some embodiments, L in formula (I) is as follows: #---mN-mN-Q304-Q304-Q304--- ** , #---dN-dN-Q304-Q304-Q304--- ** , #---rN-rN-Q304-Q304-Q304--- ** , #---rN-dN-Q304-Q304-Q304--- ** , and #---dN-rN-Q304-Q304-Q304--- ** , [In formula: dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents ribonucleotide, mN represents 2'-O-methylnucleotide. It contains one of the following.

[0060] In the above embodiment, one or more internucleotide links between nucleotides in L may independently be modified internucleotide links selected from the group consisting of phosphodiesters, phosphotryesters (which may contain a linked phosphorus atom in either the Rp or Sp configuration), hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate (which may contain a linked phosphorus atom in either the Rp or Sp configuration), phosphorothioate (which may contain a linked phosphorus atom in either the Rp or Sp configuration), and nitrogen-modified phosphorus-containing links (PN links) (which may contain a linked phosphorus atom in either the Rp or Sp configuration).

[0061] In certain embodiments, L in formula (I) is a triazole linkage, amide linkage, sulfide or disulfide linkage, phosphate linkage, oxime linkage, hydrazo linkage, N,N'-dialkylenehydrazo linkage, methyleneimino linkage, methylenecarbonylamino linkage, methylenemethylimino linkage, methylenehydrazo linkage, methylenedimethylhydrazo linkage, methyleneoxymethylimino linkage, hydroxylamino linkage, formacetal linkage, alkyl or aryl linkage, PEG linkage, ether linkage, thioether linkage, thiodiester linkage, thionocarbamate linkage, thioacetamide linkage, sulfonic acid linkage, sulfonamide linkage, sulfonic acid ester linkage, thioforma It may contain one or more linking parts selected from the group consisting of cetal linkages, urea linkages, carbonate linkages, amine linkages, maleimide-thioether linkages, phosphodiester linkages, phosphotriester linkages, hydrogen phosphonate linkages, alkyl or arylphosphonic acid linkages, phosphoramide linkages, phosphorothioate linkages, nitrogen-modified phosphorus-containing linkages (PN-linkages), phosphoroselenate linkages, boranophosphate linkages, boranophosphate ester linkages, sulfonamide linkages, carbamate linkages, carboxamide linkages, carboxymethyl linkages, carboxylate ester linkages, siloxane linkages, dialkylsiloxane linkages, ethylene oxide linkages, and combinations thereof.

[0062] In certain embodiments, L in formula (I) may contain one or more cyclic groups selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranyl, and dekalinyl.

[0063] In some embodiments, L in formula (I) contains a nucleotide-based linker (tether). In some embodiments, L contains a non-nucleotide-based linker (tether).

[0064] In certain embodiments, the nucleotide-based or non-nucleotide-based linker (tether) contained in L is a stable linker (tether) that is stable in body fluids. For example, the nucleotide-based or non-nucleotide-based stable linker (tether) is stable in plasma or artificial cerebrospinal fluid.

[0065] In a particular embodiment, the cleavage-type linking group (tether) is [ka] Formula (CL-1), and [ka] Formula (CL-2) Includes a portion selected from the group consisting of

[0066] In a particular embodiment, the cleavage-type linking group (tether) is as follows: -(CH2) 12 -(C 12 Linker or Q50), -(CH2)6-SS-(CH2)6-(C6-SS-C6 linker or Q51), Q151( [ka] ), Q173( [ka] ), -CH2CH2O-(CH2CH2) n -CH2CH2O-CH2CH2O-[wherein n is 0 or 1 to 20]; -(CH2)9-(CH2) n -CH2-[wherein n is 0 or 1 to 20]; Mono-, di-, tri-, tetra-, penta-, or polyprolinols, conjugated with ligands as appropriate. Mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinols conjugated with ligands as appropriate. Includes the portion selected from.

[0067] In certain embodiments, the tether comprises a nucleic acid linker having a length of 1 to 15 nucleotides. For example, the nucleic acid linker may have a suitably modified nucleotide length of 2 to 7, 5 to 7, 2 to 5, or 3, 4, or 5.

[0068] In certain embodiments, the cleavage linker (tether) comprises a nucleic acid linker containing one or more nucleotides selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, deoxyribonucleotide, and ribonucleotide. In one embodiment, all nucleic acid linker nucleotides are of the same type. In one embodiment, the nucleic acid linker contains entirely 2'-O-methylnucleotide, entirely 2'-fluoronucleotide, or entirely deoxyribonucleotide.

[0069] In certain embodiments, the cleavage tether comprises a polynucleotide containing a modified ribonucleotide sequence, and may also be a polynucleotide containing one or more modifications selected from the group consisting of 2'-O-methylribonucleotide modification, 2'-fluororibonucleotide modification, 2'-5'-linked nucleotides having different 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro), glycol nucleic acid (GNA) modification, roq nucleic acid (LNA) modification, hexanol nucleic acid (HNA) modification, debasalized ribose modification, debasalized deoxyribose modification, and debasalized hydroxyprolinol modification.

[0070] In some embodiments, the linking group L of the single-stranded oligonucleotide of formula (I) comprises a nucleotide-based cleavable linking group (tether) that can be cleaved by DICER. In some embodiments, the single-stranded oligonucleotide comprises a substrate that can be cleaved by DICER.

[0071] In a particular embodiment, the single-stranded oligonucleotide is a single-stranded oligonucleotide with at least one nucleotide sequence (e.g., Z 1 and / or Z 2 The compound contains a cleavable linking group (nucleotide-based or non-nucleotide-based) that can produce a metabolite of 5'-monophosphate.

[0072] In some embodiments, the single-stranded oligonucleotide may further comprise one or more ligands (e.g., targeting ligands). In one embodiment, Z 1 The sequence includes at least one ligand (e.g., a targeting ligand) at its 5' or 3' end. In one embodiment, Z 2 The sequence includes at least one ligand (e.g., a targeting ligand) at its 5' or 3' end. In one embodiment, Z 1 and Z 2 Each of these sequences contains at least one ligand (e.g., a targeting ligand) at its 5' or 3' end.

[0073] In a particular embodiment, at least one of the ligands is a lipophilic moiety.

[0074] In one embodiment, the lipophilic portion is a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, docosanic acid (DCA), dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, lithocholic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In a particular embodiment, the lipid is a fatty acid (omega-3 fatty acid, e.g.) selected from the group consisting of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0075] In some embodiments, the lipophilic portion is saturated or unsaturated C4-C 30 Hydrocarbon chains (for example, C4~C 30 It contains alkyl or alkenyl groups, as well as any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0076] In some embodiments, the lipophilic portion is saturated or unsaturated C6-C 18 Hydrocarbon chains (e.g., linear C6~C) 18 Alkyl or alkenyl compounds, for example, saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C) 16 Contains alkyl or alkenyl compounds. In some embodiments, the lipophilic portion is saturated or unsaturated C. 14 ~C 24 Hydrocarbon chains (e.g., linear C) 14 ~C 24 Alkyl or alkenyl compounds, for example, saturated or unsaturated C 22 Hydrocarbon chains (e.g., linear C) 22 It contains alkyl or alkenyl compounds. For example, one or more non-terminal positions of a single-stranded oligonucleotide have the following structure: [ka] [wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or a modified derivative thereof), and the n-hexadecyl chain is a lipophilic moiety]. The modification shown in formula (1) is referred to herein as "2'-C 16 This is called a 'continuous chain'. In another example, one or more non-terminal positions of a single-stranded oligonucleotide form the following structure: [ka] [wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or a modified derivative thereof), and the n-docosanil chain is a lipophilic moiety]. The modification shown in formula (2) is referred to herein as "2'-C 22 It is called "".

[0077] n-hexadecyl chain or n-docosanyl chain, C4~C 30 Similar modifications that substitute for hydrocarbon chains include "2'-C4~C 30 This is called a "hydrocarbon chain" (or C6~C 18 hydrocarbon chain or C 14 ~C 24 Substitution with hydrocarbon chains is "2'-C6~C 18 "Hydroxide chain" or "2'-C" 14 ~C 24 (Also known as a "hydrocarbon chain").

[0078] In related embodiments, Z 1 and Z 2 At least one or more non-terminal nucleotide positions are 2'-C4~C 30 Hydrocarbon chain structure, 2'-C6~C 18 Hydrocarbon chain structure, 2'-C 14 ~C 24 Hydrocarbon chain structure, 2'-C of formula (1) 16 Structure, or 2'-C of formula (2) 22 It has a structure.

[0079] In one embodiment, Z 1 and Z 2 Both of the one or more non-terminal nucleotide positions are 2'-C4~C 30 Hydrocarbon chain structure, 2'-C6~C 18 Hydrocarbon chain structure, 2'-C 14 ~C 24 It has a hydrocarbon chain structure, a 2'-C16 structure of formula (1), or a 2'-C22 structure of formula (2).

[0080] In some embodiments, the lipophilic portion contains one or more phospholipids.

[0081] In some embodiments, the lipophilic portion comprises one or more lipids or lipophilic ligands, all of which are disclosed in International PCT Publications WO2019 / 232255A1 and WO2021 / 108662A1, and U.S. Patent No. 10,184,124, which are incorporated herein by reference in their entirety.

[0082] In some embodiments, the ligand is expressed by the following formula: [ka] [In the formula, n is 1 in "C10-TEG-" and 7 in "C16-TEG-"] Includes one or more of the following.

[0083] In some embodiments, ligands include those disclosed in International PCT Publications WO2017 / 053999, WO2019 / 118916, WO2022 / 031433, WO2022 / 056269, WO2022 / 056273, and WO2022 / 056277, all of which are incorporated herein by reference in their entirety.

[0084] In some embodiments, Z 1 and Z 2 At least one of the nucleotide sequences includes one or more internal positions (i.e., non-terminal positions) other than positions 9–12 of the nucleotide sequence; for example, positions 4–8 and 13–18 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence as position 1; positions 5, 6, 7, 15, and 17 of the nucleotide sequence; or one or more lipophilic moieties independently conjugated to positions 4, 6, 7, and 8 of the nucleotide sequence.

[0085] In some embodiments, Z 1 and Z 2At least one of these includes one or more lipophilic moieties independently conjugated at position 6 of the nucleotide sequence, counted from the 5' end of the nucleotide sequence. In one embodiment, Z 1 and Z 2 Each of these contains a lipophilic moiety conjugated at position 6 of the nucleotide sequence; the lipophilic moiety may be saturated or unsaturated C6-C 18 Hydrocarbon chains, or saturated or unsaturated carbon. 14 ~C 24 It may also contain hydrocarbon chains; the lipophilic portion may be saturated or unsaturated C 16 Hydrocarbon chains or saturated or unsaturated carbon 22 It may contain hydrocarbon chains.

[0086] In some embodiments, Z 1 and Z 2 At least one of these includes one or more internal positions (i.e., non-terminal positions) of the nucleotide sequence; for example, positions 6-10 and 15-18 of the nucleotide sequence, counted from the 5' end of the nucleotide sequence as position 1, respectively; and one or more lipophilic moieties independently conjugated at positions 15 and 17 of the nucleotide sequence.

[0087] In some embodiments, at least one ligand is a targeting ligand selected from the group consisting of antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In one embodiment, at least one ligand is an integrin receptor ligand.

[0088] The targeting ligand is appropriately linked to a nucleotide sequence (e.g., Z) via a linker or carrier. 11 and Z 12 It can be conjugated at an internal position of ). Alternatively, the targeting ligand can be conjugated via a linker or carrier as appropriate, Z 11 and Z12 It can be conjugated to the 3' or 5' end of [the specified element].

[0089] In certain embodiments, at least one of the ligands is a carbohydrate-based ligand. Carbohydrate-based ligands may be D-galactose, polyvalent galactose, N-acetyl-D-galactosamine (GalNAc), polyvalent GalNAc, D-mannose, polyvalent mannose, polyvalent lactose, N-acetyl-glucosamine, glucose, polyvalent glucose, polyvalent fucose, glycosylated polyaminoacid, or lectin.

[0090] In certain embodiments, the carbohydrate-based ligand is an ASGPR ligand. For example, an ASGPR ligand is a divalent or trivalent branched linker, e.g.: [ka] It is one or more GalNAc derivatives joined by [the following mechanism].

[0091] In a particular embodiment, at least one ligand is a nucleotide sequence (e.g., Z 1 and Z 2 It can be conjugated to the 3' end, 5' end, or internal position of ).

[0092] In some embodiments, at least one ligand may be conjugated to a single-stranded oligonucleotide via direct linking of the oligonucleotide to a ribosugar. Alternatively, the ligand may be conjugated to the single-stranded oligonucleotide via one or more linkers (tethers) and / or carriers.

[0093] In some embodiments, the ligand may be conjugated to a single-stranded oligonucleotide via a monovalent, branched divalent, or trivalent linker.

[0094] In some embodiments, the ligand may be conjugated to a single-stranded oligonucleotide via a carrier that substitutes one or more nucleotides. The carrier may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranyl, and dekalinyl. In one embodiment, the acyclic group is a portion based on a serinol skeleton or a diethanolamine skeleton.

[0095] In some embodiments, single-stranded oligonucleotides are (a)Z 1 and Z 2 Each of these independently contains 19 to 23 appropriately modified nucleotides; (b)Q 1 and Q 2 Each of these independently contains 0 to 2 appropriately modified nucleotides; (c)Z 1 and Z 2 The double-stranded region formed by this process contains three or fewer mismatched base pairs; (d)Z 1 and Z 2 The double-stranded region formed by this process forms a blunt end; (e)Z 2 At least one nucleotide is a modified nucleotide; (f)Z 1 At least one nucleotide is a modified nucleotide; (g)Z 2 It must contain at least one modified nucleotide linkage; (h)Z 1 It must contain at least one modified nucleotide linkage; (i) The 5' terminal nucleotide contains a 5'-phosphate modification or a 5'-phosphate mimetic modification; The (j)3' terminal nucleotide may be conjugated to a ligand via a linker; (k)Z 1 This means that it contains three or fewer mismatches with respect to the target gene; (l)Z 1 and Z 2 Each independently contains 19 to 23 appropriately modified nucleotides; and (m)L is formula:#-(N) n - ** [wherein n is 3 to 5; each N independently contains a linkage represented by appropriately modified nucleotides Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, or Q368] It can be characterized by one or more of the following.

[0096] In some embodiments, single-stranded oligonucleotides may be characterized by 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all of the above features.

[0097] In some embodiments, single-stranded oligonucleotides are (a)Z 1 and Z 2 Each of these independently contains 21 appropriately modified nucleotides; (b)Q 1 and Q 2 Each of these independently contains two appropriately modified nucleotides; (c)Z 1 and Z 2 The double-stranded region formed by this process contains three or fewer mismatched base pairs; (d)Z 1 and Z 2The double-stranded region formed by this process forms a blunt end; (e)Z 2 All nucleotides are modified nucleotides; (f)Z 1 All nucleotides are modified nucleotides; (g)Z 2 It must contain at least two sequentially modified nucleotide linkages; (h)Z 1 It must contain at least two sequentially modified nucleotide linkages; (i)Z 1 The 5' terminal nucleotide contains a 5'-phosphate modification or a 5'-phosphate mimetic modification; (j)Z 2 The 3' terminal nucleotide may be conjugated to a ligand via a linker; (k)Z 1 This means that it contains three or fewer mismatches with respect to the target gene; and (l)L is the formula:#-(N) n - ** The formula contains a linkage represented by [wherein n is 5; each N is independently a appropriately modified nucleotide or Q304] It can be characterized by one or more of the following.

[0098] In some embodiments, single-stranded oligonucleotides may be characterized by 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all of the above features.

[0099] Another aspect of the present invention is formula (II) or (III): (5'-Z 11 -3')-LQ S -(5'-Z 12 -3') (II), (3'-Z 11 -5')-LQ S-(3'-Z 12 -5') (III), [In formula: Z 11 This is a first oligonucleotide containing 15 to 100 appropriately modified nucleotides that are substantially complementary to the target gene; Z 12 is, Z 11 A second oligonucleotide containing 10 to 100 appropriately modified nucleotides, substantially complementary to the first oligonucleotide; Z 11 and Z 12 This can form an intrachain double-strand region containing 7 or more consecutive base pairs; Q S This represents appropriately modified nucleotides from 0 to 12. L is any linking group. A single-stranded oligonucleotide by At least one nucleotide in formula (II) is a modified nucleotide; and At least one nucleotide in formula (III) is a modified nucleotide, where, In equation (II), Z 11 At least one nucleotide at the 3' end of formula (III), Z 11 At least one nucleotide at the 5' end is L and Q in both cases. S Along with Z 11 and Z 12 Forms a loop region that connects Regarding single-stranded oligonucleotides.

[0100] In some embodiments, the single-stranded oligonucleotide may be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimir (antagomir) oligonucleotide, a microRNA mimetic, a supermir, an aptamer, a U1 adapter, a triple-stranded oligonucleotide, an RNA activator, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a heterodouble-stranded oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide.

[0101] First oligonucleotide Z 11 and the second oligonucleotide Z 12 Each of these can independently contain 10 to 100 appropriately modified nucleotides. For example, Z 11 and Z 12 Each independently comprises appropriately modified nucleotides of 10-40, 10-30, 12-26, 12-23, 12-21, 15-26, 15-23, 15-21, 19-26, 19-23, or 19-21. In some embodiments, the first oligonucleotide Z 11 and the second oligonucleotide Z 12 Each of these may independently contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotide lengths. 11 and Z 12 These are, independently, approximately 10-50 nucleotides, approximately 10-40 nucleotides, approximately 10-35 nucleotides, approximately 10-30 nucleotides, approximately 10-26 nucleotides, approximately 10-23 nucleotides, approximately 10-21 nucleotides, approximately 12-50 nucleotides, approximately 12-40 nucleotides, approximately 12-35 nucleotides, approximately 12-30 nucleotides, approximately 12-26 nucleotides, approximately 12-23 nucleotides, approximately 12-21 nucleotides, and approximately 15-5 They may have a length of 0 nucleotides, approximately 15 to 40 nucleotides, approximately 15 to 35 nucleotides, approximately 15 to 30 nucleotides, approximately 15 to 26 nucleotides, approximately 15 to 23 nucleotides, approximately 15 to 21 nucleotides, approximately 19 to 50 nucleotides, approximately 19 to 40 nucleotides, approximately 19 to 35 nucleotides, approximately 19 to 30 nucleotides, approximately 19 to 26 nucleotides, approximately 19 to 23 nucleotides, approximately 19 to 21 nucleotides, or approximately 18 to 20 nucleotides.

[0102] In some embodiments, Z 11and Z 12 Each independently contains 10 to 40 appropriately modified nucleotides. In some embodiments, Z 11 and Z 12 Each of these independently contains 12 to 26 appropriately modified nucleotides.

[0103] In some embodiments, Z 11 and Z 12 Each contains the same number of appropriately modified nucleotides. In some embodiments, Z 11 is, Z 12 It contains more appropriately modified nucleotides. In some embodiments, Z 11 It contains 19-26 appropriately modified nucleotides, Z 12 It contains 12 to 21 appropriately modified nucleotides.

[0104] In some embodiments, a single-stranded oligonucleotide can be cleaved at a linking group L. First oligonucleotide Z 11 It is cleaved into an antisense strand that is substantially complementary to the target gene (e.g., target mRNA or DNA), and a second oligonucleotide Z 12 is, Z 11 It can be cut into a sense chain that is substantially complementary to it.

[0105] Q S It may contain appropriately modified nucleotides from 0 to 12. For example, Q S This may include appropriately modified nucleotides of 0-10, 0-6, 0-4, 0-3, 0-2, 1-6, 1-4, 1-3, 1-2, or 2-3. In some embodiments, Q S Q is 0. In some embodiments, Q S is a suitably modified nucleotide from 1 to 6. In some embodiments, Q S is a suitably modified nucleotide of 2. In some embodiments, Q S This is a nucleotide with appropriate modifications to 1.

[0106] In some embodiments, Q S One or more nucleotides of Z 11 It forms a mismatch base pair with the opposite nucleotide. In some embodiments, Q S is a modified nucleotide of 2, as follows: Q S Both nucleotides are Z 11 Forming mismatch base pairs with their opposite nucleotides Q S One of its nucleotides is Z 11 It forms a mismatch base pair with the opposite nucleotide (for example, Z 12 Q next to S The nucleotide is Z 11 (to form mismatch base pairs with those nucleotides opposite to them), or Q S Both nucleotides are Z 11 To form a base pair with the opposite nucleotide of those nucleotides. It is characterized by one of the following.

[0107] In some embodiments, Q S Q is two 2'-deoxy-modified nucleotides. In some embodiments, Q S It is -dTdT-.

[0108] First oligonucleotide Z 11 It is substantially complementary to the target gene, namely, Z 11 The target gene contains a mismatch of 3 or fewer (e.g., 0, 1, 2, or 3). In some embodiments, the target gene may be mRNA, pre-mRNA, microRNA, pre-miRNA, long non-coding RNA (lncRNA), or DNA.

[0109] First oligonucleotide Z 11 and the second oligonucleotide Z 12 For example, it can form an intrachain double-strand region containing 7 or more consecutive base pairs. In some embodiments, Z11 and Z 12 This can form an intrachain double-strand region containing 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 base pairs. In some embodiments, Z 11 and Z 12 is, Z 12 It can form an intrachain double-strand region having base pairs with all of the nucleotides. 11 and Z 12 The intrachain double helix region formed by this may contain all consecutive base pairs or up to 3 mismatched base pairs (e.g., 0, 1, 2, or 3). In some embodiments, Z 11 and Z 12 The intrachain double-strand region formed by this process contains one mismatched base pair.

[0110] In some embodiments, the first oligonucleotide Z 11 and the second oligonucleotide Z 12 is, Z 11 An intrachain double helix region can be formed in the seed region (e.g., the seed region of the antisense chain; e.g., at positions 2-8 of the 5' end of the antisense chain).

[0111] In some embodiments, the first oligonucleotide Z 11 It contains a loop at the 3' or 5' end. In some embodiments, the first oligonucleotide Z 11 In the formula W-LP[wherein W is Z], 12 It can form an intrachain double helix region of at least 7 base pairs, and LP, along with L as appropriate, has W and Z at the 3' or 5' end. 12 This includes forming a loop between W and Z in some embodiments. 12These can form an intrachain double-strand region containing 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 base pairs. In some embodiments, W and Z 12 is, Z 12 It is possible to form an intrachain double-strand region having base pairs with all nucleotides of W and Z. 12 The intrachain double-strand region formed by this may contain all consecutive base pairs or three or fewer mismatched base pairs (e.g., 0, 1, 2, or 3).

[0112] In some embodiments, the single-stranded oligonucleotide is defined as formula (IIa) or formula (IIIa): [ka] [In formula: Z 11 This includes W-LP, W is Z 12 This forms an intrachain double helix region of at least 7 base pairs, LP, along with L as appropriate, has W and Z at its 3' or 5' end. 12 Forming a loop between them, [ka] This represents any existence of L, [ka] Q S Represents any existence of, [ka] is, Z 11 Represents any overhang at the 5' or 3' end of the object. [ka] is, Z 12[Represents any overhang at the 5' or 3' end] It is represented by [this].

[0113] In some embodiments, Z at the non-loop end 11 and Z 12 The double-stranded region formed by has blunt ends. In some embodiments, W and Z are present at the non-loop ends. 12 The double-stranded region formed by this process has a blunt end.

[0114] In some embodiments, Z at the non-loop end 11 It has an overhang of 1 to 3 nucleotides in length. In some embodiments, W at the non-loop end has an overhang of 1 to 3 nucleotides in length. In some embodiments, [ka] These exist and are 1 to 3 nucleotides long.

[0115] In some embodiments, Z 12 It has an overhang of 1 to 3 nucleotides in length. In some embodiments, [ka] These exist and are 1 to 3 nucleotides long.

[0116] In some embodiments, the overhang is 1 nucleotide long. In some embodiments, the overhang is 2 nucleotides long. In some embodiments, the overhang is 3 nucleotides long.

[0117] Each nucleotide within a single-stranded oligonucleotide can be modified independently and as appropriate. First oligonucleotide Z 11 and the second oligonucleotide Z 12 Each of the nucleotides within can be modified independently and as appropriate.

[0118] In some embodiments, the single-stranded oligonucleotide includes at least one chemical modification. In some embodiments, the first oligonucleotide Z 11 and the second oligonucleotide Z 12 Each of these includes at least one chemical modification. In some embodiments, W includes at least one chemical modification. In some embodiments, Z 11 All nucleotides of are modified nucleotides. In some embodiments, all nucleotides of W are modified nucleotides. In some embodiments, Z 12 All of the nucleotides are modified nucleotides. In some embodiments, all nucleotides of a single-stranded oligonucleotide are modified.

[0119] Chemical modifications to nucleotides may include nucleoside linkage modifications, nucleobase modifications, sugar modifications, or combinations thereof.

[0120] In certain embodiments, the chemical modification is selected from the group consisting of LNA, ENA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modification (e.g., 2'-modified L-nucleoside, e.g., 2'-deoxy-L-nucleoside), BNA debasic sugars, debasic cyclic and open-chain alkyls, and combinations thereof.

[0121] In certain embodiments, the chemical modification is selected from the group consisting of at least one modified nucleotide, and includes deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, lock nucleotides (LNAs), unlock nucleotides (UNAs), structurally restricted nucleotides, restricted ethyl nucleotides, debasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramides, non-natural bases containing nucleotides, and tetrahydropyran modifications. These are nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing 5'-phosphate modification or a 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleotides containing glycol nucleic acid (GNA), nucleotides containing glycol nucleic acid (GNA) S isomers (S-GNA), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-5'-linked nucleotides ("3'-RNA"), or terminal nucleotides linked to cholesteryl derivatives or dodecanoate bisdecylamide groups.

[0122] In certain embodiments, the chemical modification is a 2'-modification selected from the group consisting of 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, 2'-fluoro, and combinations thereof.

[0123] In some embodiments, Z is approximately 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30%. 11 Z is modified. In some embodiments, Z is approximately 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30%. 12 The nucleotides are modified. In some embodiments, about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of all nucleotides of the single-stranded oligonucleotide are modified. For example, if 50% of all nucleotides are modified, then 50% of all nucleotides present in the single-stranded oligonucleotide contain at least one modification described herein.

[0124] In one embodiment, at least 50% of the nucleotides of a single-stranded oligonucleotide are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, or 2'-fluoro.

[0125] In some embodiments, a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one or more of the following internucleotide ligation modifications: (i) One or more internucleotide links within the six 3' terminal nucleotides are modified internucleotide links; and (ii) One or more internucleotide links within the six 5' terminal nucleotides are modified internucleotide links.

[0126] In some embodiments, a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) is Z of formula (II). 11It further comprises one or more internucleotide links within the eight 3' terminal nucleotides of formula (III), or Z 11 One or more internucleotide junctions within the eight 5' terminal nucleotides are modified internucleotide junctions.

[0127] In some embodiments, a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one or more of the following internucleotide ligation modifications: (i) Two consecutive nucleotide junctions within six 3' terminal nucleotides are modified nucleotide junctions; and (ii) Two consecutive nucleotide links within the six 5' terminal nucleotides are modified nucleotide links.

[0128] In some embodiments, if a single-stranded oligonucleotide contains a ligand terminal conjugation to the 5' or 3' terminal nucleotide, or contains a debasalized nucleotide, an inverted nucleotide, or an inverted debasalized nucleotide terminal conjugation, then the above-mentioned internucleotide ligation modification to the terminal nucleotide may be removed at its end.

[0129] In some embodiments, a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one of the following internucleotide ligation modifications: (i) Z of equation (II) (or IIa) 11 One or more internucleotide links within the eight 3' terminal nucleotides, or Z of formula (III) (or (IIIa)) 11 One or more internucleotide junctions within the eight 5' terminal nucleotides are modified internucleotide junctions; (ii) Z of equation (II) (or IIa) 11 One or more internucleotide links within the six 5' terminal nucleotides, or Z of formula (III) (or (IIIa)) 11One or more internucleotide junctions within the six 3' terminal nucleotides are modified internucleotide junctions; (iii) Z of equation (II) (or (IIa)) 12 One or more internucleotide links within the six 5' terminal nucleotides, or Z of formula (III) (or (IIIa)) 12 One or more internucleotide junctions within the six 3' terminal nucleotides are modified internucleotide junctions; and (iv) Z of equation (II) (or (IIa)) 12 One or more internucleotide links within the six 3' terminal nucleotides, or Z of formula (III) (or (IIIa)) 12 One or more internucleotide junctions within the six 5' terminal nucleotides are modified internucleotide junctions.

[0130] In some embodiments, a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one or more of the following internucleotide ligation modifications: (i) Z of equation (II) (or (IIa)) 12 The linkage between two consecutive nucleotides within the three 5' terminal nucleotides, or the Z of formula (III) (or (IIIa)). 12 Two consecutive nucleotide junctions within the three 3' terminal nucleotides are modified nucleotide junctions; (ii) Z of equation (II) (or (IIa)) 12 The linkage between two consecutive nucleotides within the three 3' terminal nucleotides, or the Z of formula (III) (or (IIIa)). 12 The two consecutive nucleotide junctions within the three 5' terminal nucleotides are modified nucleotide junctions; and (iii) Z of equation (II) (or (IIa)) 11 A linkage between two consecutive nucleotides within three or four 5' terminal nucleotides, or Z of formula (III) (or (IIIa)). 11Two consecutive nucleotide junctions within three or four 3' terminal nucleotides are modified nucleotide junctions.

[0131] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) is Z 12 The 5' terminal nucleotide and Q S The first nucleotide further comprises one or more modified nucleotide linkages. In some embodiments, the single-stranded oligonucleotide of formula (III) (or (IIIa)) is Z 12 The 3' terminal nucleotide and Q S The first nucleotide further comprises one or more modified internucleotide links. In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or IIIa) is Q S The nucleotides further include one or more modified internucleotide links.

[0132] In some embodiments, and in all of the above embodiments, the modified nucleotide linkage is a phosphorothioate linkage.

[0133] In some embodiments, a single-stranded oligonucleotide of formula (II) (or (IIa)) or formula (III) (or (IIIa)) comprises at least two sequential phosphorothioate or methylphosphonate nucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least two blocks of two sequential phosphorothioate or methylphosphonate nucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least three blocks of two sequential phosphorothioate or methylphosphonate nucleotide linkage modifications.

[0134] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) is a nucleotide sequence (e.g., Z 11 and / or Z 12It has at least two phosphorothioate nucleotide links within the first six nucleotides.

[0135] In some embodiments, single-stranded oligonucleotides (e.g., Z 11 and / or Z 12 The nucleotide sequence of ) contains two blocks of one, two, or three phosphorothioate nucleotide links separated by phosphate nucleotide links of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0136] In one embodiment, the nucleotide sequence of a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) (for example, Z 11 and / or Z 12 The nucleotide sequence, counting from the 5' end of the nucleotide sequence, contains at least two consecutive phosphorothioate nucleotide linkage modifications within positions 18-23 of the nucleotide sequence. In one embodiment, the nucleotide sequence of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) contains at least two consecutive phosphorothioate nucleotide ligation modifications within the 1-5 position of the nucleotide sequence, counted from the 5' end of the nucleotide sequence.

[0137] In some embodiments, the Z of a single-stranded oligonucleotide 11 and Z 12 Each of these includes at least two consecutive phosphorothioate nucleotide ligation modifications. In one embodiment, the Z of a single-strand oligonucleotide 11 and Z 12 Each of these includes at least two consecutive phosphorothioate internucleotide ligation modifications within positions 18–23 of the nucleotide sequence, and at least two consecutive phosphorothioate internucleotide ligation modifications within positions 1–5 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence.

[0138] In some embodiments, Z at the non-loop end 11 It has an overhang of 1 to 3 nucleotides in length. In one embodiment, Z at the non-loop end 11 It has an overhang of 2 nucleotides in length (for example, Z 11 (at the 3' end), it has phosphorothioate nucleotide linkages between the two overhangs. In one embodiment, Z at the non-loop end 11 It has an overhang of 2 nucleotides in length and has two phosphorothioate nucleotide links between the terminal 3 nucleotides (for example, Z 11 At the 3' end, two of the three nucleotides are overhanging nucleotides, and the third is the next pairing nucleotide of the overhanging nucleotide.

[0139] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) is a nucleotide sequence (e.g., Z 11 and / or Z 12 ) further comprises a phosphate group or a phosphate mimetic at the 5' end. In some embodiments, the single-stranded oligonucleotide has a nucleotide sequence (e.g., Z 11 and / or Z 12 ) contains a phosphate mimetic at its 5' end. In one embodiment, at least one phosphate mimetic is Z 11 It is located at the 5' end. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP). In one embodiment, the phosphate mimetic is 5'-cyclopropylphosphonate. In one embodiment, the phosphate mimetic is 5'-vinylphosphate.

[0140] In some embodiments, the single-chain oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises at least one terminal, a chiral phosphorus atom.

[0141] In some embodiments, the 5' or 3' terminal nucleotide of a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) includes a 2'-5'-linked nucleotide modification, or the 5' or 3' terminal nucleotide is conjugated to a debasalized nucleotide, inverted nucleotide, or inverted debasalized nucleotide (e.g., ribonucleotide) via a phosphodiester, phosphorothioate, or phosphodithioate linkage, as appropriate.

[0142] In some embodiments, the 5' or 3' terminal nucleotide of a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) is modified and includes a linking moiety containing mono-, di-, tri-, tetra-, penta-, or polyprolinol, or mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinol.

[0143] In some embodiments, single-stranded oligonucleotides (e.g., Z 11 and / or Z 12 Each of the at least one, two, three, four, or five terminal phosphorus-containing links at the 5' or 3' end of ) is not a phosphorothioate linkage. In one embodiment, a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 Each of the at least one, two, three, four, or five terminal phosphorus-containing linkages at the 5' or 3' end of the ) is independently a natural phosphate group or phosphodiester linkage, or a PN-linkage.

[0144] In some embodiments, single-stranded oligonucleotides (e.g., Z 11 and / or Z 12Each of the at least one, two, three, four, or five terminal phosphorus-containing links at the 5' or 3' end of ) is -N(R)P(=X)(OH)O- or -OP(=X)(OH)N(R)-, -OP(NR)(=X)O-, N(SO2R)P(=X)(OH)O- or -OP(=X)(OH)N(SO2R)-, or -OP(NSO2R)(=X)O- [wherein X is O or S; R may be an optionally substituted alkyl, aryl, heteroaryl or heterocyclyl; or NR may be an optionally substituted cyclic guanidine moiety, an optionally substituted triazolyl group or Tmg group] [ka] It is a PN connection having the expression [which can be].

[0145] In some embodiments, single-stranded oligonucleotides (e.g., Z 11 and / or Z 12 Each of the at least one, two, three, four, or five terminal phosphorus-containing links at the 5' or 3' end of ) is appropriately substituted with a cyclic guanidine moiety, for example, [ka] This is a PN linkage having the structure [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S.

[0146] In some embodiments, single-stranded oligonucleotides (e.g., Z 11 and / or Z 12 Each of the at least one, two, three, four, or five terminal phosphorus-containing links at the 5' or 3' end of ) is a triazole moiety (e.g., appropriately substituted triazole groups), e.g., [ka] This is a PN linkage including those having the structure [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S.

[0147] In some embodiments, single-stranded oligonucleotides (e.g., Z 11 and / or Z 12 Each of the at least one, two, three, four, or five terminal phosphorus-containing links at the 5' or 3' end of ) is an alkyne moiety (e.g., an appropriately substituted alkynyl group), for example, [ka] This is a PN linkage including those having the structure [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S.

[0148] In some embodiments, single-stranded oligonucleotides (e.g., Z 11 and / or Z 12 Each of the at least one, two, three, four, or five terminal phosphorus-containing links at the 5' or 3' end of ) is a Tmg group [ka] ,for example, [ka] This is a PN linkage including those having the structure [wherein W is O or S]. In some embodiments, W is O. In some embodiments, W is S.

[0149] In all of the above embodiments, PN linkage can be stereochemically controlled.

[0150] In some embodiments, in a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), L, Q S , or Z 12 Connected, Z 11The 3 to 5 terminal nucleotides contain modifications selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-ara nucleotide (aN).

[0151] In some embodiments, in a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), L, Q S , or Z 12 Connected, Z 11 The three terminal nucleotides are, #-fN-fN-fN- ** , #-dN-dN-dN- ** , #-dN-dN-rN- ** , #-dN-rN-dN- ** , #-rN-dN-dN- ** , #-rN-rN-dN- ** , #-rN-dN-rN- ** , #-dN-rN-rN- ** , and #-rN-rN-rN- ** [In formula: # is Z 11 It is a coupling to, ** L, Q S , or Z 12 It is a coupling to dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents ribonucleotide, mN represents 2'-O-methylnucleotide. It has modifications selected from the group consisting of the following.

[0152] In some embodiments, in a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), L, Q S , or Z 12 Connected, Z 11 The five terminal nucleotides are, #-dN-dN-fN-fN-fN- ** , #-dN-dN-rN-dN-dN- ** , #-dN-dN-rN-rN-rN- ** , #-dN-dN-dN-dN-dN- ** , #-mN-mN-fN-fN-fN- ** , #-mN-mN-dN-dN-dN- ** , #-mN-mN-rN-dN-dN- ** , #-mN-mN-rN-rN-rN- ** [In formula: # is Z 11 It is a coupling to, ** L, Q S , or Z 12 It is a coupling to dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents ribonucleotide, mN represents 2'-O-methylnucleotide. It has modifications selected from the group consisting of the following.

[0153] In some embodiments, in a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), LP and / or L are L and / or Q S Together with Z 12 The connected items are #-dN-dN-rN-rN-rN-rN-dN- ** [In formula: # is Z11 It is a coupling to, ** is, Z 12 It is a coupling to, dN represents a 2'-deoxynucleotide, and rN represents a ribonucleotide (e.g., uridine or 5-methyluridine). It includes the following modification.

[0154] In some embodiments, in a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), LP and / or L are L and / or Q S Together with Z 12 The connected items are #-dT-dT-rN-rN-rN-dN-dN- ** [In formula: # is Z 11 It is a coupling to, ** is, Z 12 It is a coupling to, dN represents a 2'-deoxynucleotide, and rN represents a ribonucleotide (e.g., uridine or 5-methyluridine). It includes the following modification.

[0155] In some embodiments, in a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), LP and / or L are L and / or Q S Together with Z 12 The connected items are #-dN-dN-rN-rN-rN-dT-dT- ** [In formula: # is Z 11 It is a coupling to, ** is, Z 12 It is a coupling to, dN represents a 2'-deoxynucleotide, and rN represents a ribonucleotide (e.g., uridine or 5-methyluridine). It includes the following modification.

[0156] In some embodiments, in a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), LP and / or L are L and / or Q S Together with Z 12 The connected items are #-dT-dT-rN-rN-rN-dT-dT- ** [In formula: # is Z 11 It is a coupling to, ** is, Z 12 It is a coupling to, dN represents a 2'-deoxynucleotide, and rN represents a ribonucleotide (e.g., uridine or 5-methyluridine). It includes the following modification.

[0157] In some embodiments, the first oligonucleotide Z 11 is, Z 11 It contains at least one motif of three consecutive 2'-O-methyl modifications at positions 11, 12, and 13 from the 5' end, and the nucleotide following the motif is not 2'-O-methyl modified.

[0158] In some embodiments, the second oligonucleotide Z 12 Q as appropriate S It also contains at least one motif of three consecutive 2'-F modifications, and the nucleotide following the motif is not 2'-F modified.

[0159] In some embodiments, the positions of the three consecutive modifications (three consecutive 2'-O-methyl modifications or three consecutive 2'-F modifications) are as follows: The motif is Q S , Z 12 They are in 1st and 2nd place, Z 11 It may be 19 nucleotides long; The motif is Z 12 It is in 1st, 2nd, and 3rd place, Z 11 It may be 20 nucleotides long; The motif is Z12 They are in 2nd, 3rd and 4th place, Z 11 It may be 21 nucleotides long; The motif is Z 12 It is in 3rd, 4th and 5th place, Z 11 It may be 22 nucleotides long; or The motif is Z 12 It is in 4th, 5th and 6th place, Z 11 It may be 23 nucleotides long; It is characterized by one of the following.

[0160] In some embodiments, Z 12 Q as appropriate S Along with that, its position is Z 11 Unless it is part of the motif, it contains a 2'-O-methyl or 2'-F modification at the position two positions before the motif (n-2 position if the motif is at position n).

[0161] In some embodiments, L is a cleavage linker. In some embodiments, the cleavage linker is cleavable in homogenates, tritosomes, cytosols, or endosomes of any type of cell. For example, the cleavage linker may be cleavable in liver homogenates, liver tritosomes, liver lysosomes, liver cytosols, liver endosomes, brain homogenates, brain tritosomes, brain lysosomes, brain cytosols, or brain endosomes. In certain embodiments, the cleavage linker is a redox cleavage linker (e.g., a reductive cleavage linker; e.g., a disulfide group), an acid cleavage linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavage linker (e.g., an ester group), a phosphatase cleavage linker (e.g., an ester group), a peptidase cleavage linker (e.g., an ester group), or an endosome cleavage linker (or a protease cleavage linker, e.g., a carbohydrate linker).

[0162] In some embodiments, the cleavage linker (tether) is an endosomal cleavage linker or a protease cleavage linker, such as a carbohydrate linker, which is cleaved at least 1.25 times 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).

[0163] In some embodiments, L exists in formula (II) (or IIa) or formula III (or IIIa), and formula:#-(N) n - ** It contains a connecting part represented by . In this formula, # is Z 11 It is a coupling to, ** Q S or Z 12 The bond is to; n is 3 to 12; each N is independently a linked monomer having a chain length of 3 or more atoms. For example, each N may independently be a linked monomer having a chain length of 3 or more atoms. "Chain length" is defined herein as above. In some embodiments, n is 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5. In one embodiment, n is 3.

[0164] In some embodiments, one or more linkages (N) in L of formula (II) (or IIa) or formula III (or IIIa) may be appropriately modified nucleotides.

[0165] In some embodiments, one or more linkages (N) in L of formula (II) (or IIa) or formula III (or IIIa) may be independently selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide).

[0166] In certain embodiments, one or more linking portions (N) in L of formula (II) (or IIa) or formula III (or IIIa) are phosphodiesters, phosphotryesters (which may contain a linked phosphorus atom in either the Rp or Sp configuration), hydrogen phosphonates, alkyl or aryl phosphonates, phosphoramidates (which may contain a linked phosphorus atom in either the Rp or Sp configuration), phosphorothioates (which may contain a linked phosphorus atom in either the Rp or Sp configuration), methylene methyliminos, nitrogen-modified phosphorus-containing linkages (PN linkages) (which may contain a linked phosphorus atom in either the Rp or Sp configuration), thiodiesters, thionocarbamates, It may contain modified nucleotide linkages selected from the group consisting of N,N'-dimethylhydrazine, phosphoroselenates, boranophosphates, boranophosphate esters, amides, hydroxylaminos, siloxanes, dialkylsiloxanes, carboxamides, carbonates, carboxymethyls, carbamates, carboxylate esters, thioethers, ethylene oxide linkers, sulfides, sulfonates, sulfonamides, sulfonate esters, thioformacetals, formacetals, oximes, methyleneiminos, methylenecarbonylaminos, methylenehydrazos, methylenedimethylhydrazos, methyleneoxymethyliminos, ethers, thioacetamides, and combinations thereof.

[0167] In certain embodiments, one or more linking portions (N) in L of formula (II) (or IIa) or formula III (or IIIa) may contain a portion selected from the group consisting of: aliphatic saturated or unsaturated alkyl chains; phosphorus-containing links including phosphoric acid, phosphonates, phosphoramidates (which may contain linked phosphorus atoms in either the Rp or Sp configuration), phosphodiesters, phosphotryesters (which may contain linked phosphorus atoms in either the Rp or Sp configuration), phosphorothioates (which may contain linked phosphorus atoms in either the Rp or Sp configuration), and nitrogen-modified phosphorus-containing links (PN links) (which may contain linked phosphorus atoms in either the Rp or Sp configuration); (poly)ethylene glycol chains including diethylene glycol, triethylene glycol, tetra, penta, hexa, hepta, octa, nona, or decaethylene glycol; glycerol or glycerol esters; aminoalkyl ethers; and combinations thereof.

[0168] In some embodiments, one or more linking portions (N) in L of formula (II) (or IIa) or formula III (or IIIa) may contain portions selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, as well as combinations thereof.

[0169] In some embodiments, one or more connecting portions (N) in L of formula (II) (or IIa) or formula III (or IIIa) are independently, [ka] [In formula: Base is a nucleobase that has been modified as appropriate. R D C4~ 30 Alkyl, C4~ 30 Alkenyl, or C4~ 30 [It is Alkinnil] A selection can be made from the group consisting of the following.

[0170] In some embodiments, one or more linkages (N) in L of formula (II) (or IIa) or formula III (or IIIa) include mono-, di-, tri-, tetra-, penta-, or polyprolinol appropriately conjugated with a ligand; mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinol appropriately conjugated with a ligand; appropriately modified nucleotides; or combinations thereof.

[0171] In some embodiments, L in formula (II) (or IIa) or formula III (or IIIa) comprises one or more mono-, di-, tri-, tetra-, penta-, or polyprolinols optionally conjugated with a ligand; and one or more optionally modified nucleotides.

[0172] In some embodiments, L in formula (II) (or IIa) or formula III (or IIIa) comprises one or more mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinols, optionally conjugated with a ligand; and one or more optionally modified nucleotides.

[0173] In some embodiments, one or more connecting portions (N) in L of formula (II) (or IIa) or formula III (or IIIa) may be independently selected from the group consisting of Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, and Q368.

[0174] In some embodiments, each linkage (N) in L of formula (II) (or IIa) or formula III (or IIIa) is independently a suitably modified nucleotide, Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, or Q368.

[0175] In some embodiments, L in formula (II) (or IIa) or formula III (or IIIa) contains 3 to 5 2'-deoxynucleotides, a triplet of 2'-deoxy-2'-fluoronucleotides, a triplet of ribonucleotides, a triplet of 2'-O-methylnucleotides, or a triplet of Q304. In one embodiment, L contains a triplet of Q304.

[0176] In some embodiments, the position of L in formula (II) (or IIa) or formula III (or IIIa) is as follows: All linked monomers of L, together with LP, are W and Z. 12 Forming a loop between them; One or more linked monomers of L, together with LP, W and Z 12 A loop is formed between them, and one or more linked monomers of L are not in the loop region; One or more linked monomers of L, together with LP, W and Z 12 A loop is formed between them, and one or more linked monomers of L are not in the loop, Q S Being connected; and One or more linked monomers of L, together with LP, W and Z 12 A loop is formed between them, and one or more linked monomers of L are not in the loop, Z 12 Being connected It is characterized by one of the following.

[0177] In the embodiments described above, one or more internucleotide links between nucleotides in L of formula (II) (or IIa) or formula III (or IIIa) may independently be modified internucleotide links selected from the group consisting of phosphodiesters, phosphotryesters (which may contain a linked phosphorus atom in either the Rp or Sp configuration), hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate (which may contain a linked phosphorus atom in either the Rp or Sp configuration), phosphorothioate (which may contain a linked phosphorus atom in either the Rp or Sp configuration), and nitrogen-modified phosphorus-containing links (PN links) (which may contain a linked phosphorus atom in either the Rp or Sp configuration).

[0178] In certain embodiments, L in formula (II) (or IIa) or formula III (or IIIa) is a triazole linkage, amide linkage, sulfide or disulfide linkage, phosphate linkage, oxime linkage, hydrazo linkage, N,N'-dialkylenehydrazo linkage, methyleneimino linkage, methylenecarbonylamino linkage, methylenemethylimino linkage, methylenehydrazo linkage, methylenedimethylhydrazo linkage, methyleneoxymethylimino linkage, hydroxylamino linkage, formacetal linkage, alkyl or aryl linkage, PEG linkage, ether linkage, thioether linkage, thiodiester linkage, thionocarbamate linkage, thioacetamide linkage, sulfonic acid linkage, sulfonamide linkage, sulfone It may contain one or more linking moieties selected from the group consisting of acid ester linkages, thioform acetal linkages, urea linkages, carbonate linkages, amine linkages, maleimide-thioether linkages, phosphodiester linkages, phosphotriester linkages, hydrogen phosphonate linkages, alkyl or arylphosphonic acid linkages, phosphoramide linkages, phosphorothioate linkages, nitrogen-modified phosphorus-containing linkages (PN-linkages), phosphoroselenate linkages, boranophosphate linkages, boranophosphate ester linkages, sulfonamide linkages, carbamate linkages, carboxamide linkages, carboxymethyl linkages, carboxylate ester linkages, siloxane linkages, dialkylsiloxane linkages, ethylene oxide linkages, and combinations thereof.

[0179] In certain embodiments, L in formula (II) (or IIa) or formula III (or IIIa) may contain one or more cyclic groups selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranyl, and dekalinyl.

[0180] In some embodiments, L in formula (II) (or IIa) or formula III (or IIIa) contains a nucleotide-based linker (tether). In some embodiments, L contains a non-nucleotide-based linker (tether).

[0181] In certain embodiments, the nucleotide-based or non-nucleotide-based linker (tether) contained in L of formula (II) (or IIa) or formula III (or IIIa) is a stable linker (tether) that is stable in body fluids. For example, the nucleotide-based or non-nucleotide-based stable linker (tether) is stable in plasma or artificial cerebrospinal fluid.

[0182] In a particular embodiment, the cleavage-type linking group (tether) includes a portion of formula (CL-1) or (CL-2) as described above.

[0183] In a particular embodiment, the cleavage-type linking group (tether) is as follows: -(CH2) 12 -(C12 linker or Q50), -(CH2)6-SS-(CH2)6-(C6-SS-C6 linker or Q51), Q151, Q173, -CH2CH2O-(CH2CH2) n-CH2CH2O-CH2CH2O-[wherein n is 0 or 1 to 20]; -(CH2)9-(CH2) n -CH2-[wherein n is 0 or 1 to 20]; Mono-, di-, tri-, tetra-, penta-, or polyprolinols, conjugated with ligands as appropriate. Mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinols conjugated with ligands as appropriate. Includes the portion selected from.

[0184] In certain embodiments, the tether comprises a nucleic acid linker having a length of 1 to 15 nucleotides. For example, the nucleic acid linker may have a suitably modified nucleotide length of 2 to 7, 5 to 7, 2 to 5, or 3, 4, or 5.

[0185] In certain embodiments, the cleavage linker (tether) comprises a nucleic acid linker containing one or more nucleotides selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, deoxyribonucleotide, and ribonucleotide. In one embodiment, all nucleic acid linker nucleotides are of the same type. In one embodiment, the nucleic acid linker contains entirely 2'-O-methylnucleotide, entirely 2'-fluoronucleotide, or entirely deoxyribonucleotide.

[0186] In certain embodiments, the cleavage tether comprises a polynucleotide containing a modified ribonucleotide sequence, and may also be a polynucleotide containing one or more modifications selected from the group consisting of 2'-O-methylribonucleotide modification, 2'-fluororibonucleotide modification, 2'-5'-linked nucleotides having different 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro), glycol nucleic acid (GNA) modification, roq nucleic acid (LNA) modification, hexanol nucleic acid (HNA) modification, debasalized ribose modification, debasalized deoxyribose modification, and debasalized hydroxyprolinol modification.

[0187] In some embodiments, the linking group L of the single-stranded oligonucleotide of formula (II) (or IIa) or formula III (or IIIa) includes a nucleotide-based cleavable linking group (tether) that can be cleaved by DICER. In some embodiments, the single-stranded oligonucleotide includes a substrate that can be cleaved by DICER.

[0188] In a particular embodiment, a single-stranded oligonucleotide of formula (II) (or IIa) or formula III (or IIIa) is a single-stranded oligonucleotide of which at least one nucleotide sequence (e.g., Z 11 and / or Z 12 The compound contains a cleavable linking group (nucleotide-based or non-nucleotide-based) that can produce a metabolite of 5'-monophosphate.

[0189] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula III (or IIIa) may further comprise one or more ligands (e.g., targeting ligands). In one embodiment, Z 11 The sequence includes at least one ligand (e.g., a targeting ligand) at its 5' or 3' end. In one embodiment, Z 12 The sequence includes at least one ligand (e.g., a targeting ligand) at its 5' or 3' end. In one embodiment, Z 11 and Z 12 Each of these sequences contains at least one ligand (e.g., a targeting ligand) at its 5' or 3' end.

[0190] In some embodiments, at least one ligand is optionally linked to a nucleotide sequence (e.g., Z) via a linker or carrier. 11 and Z 12 It is conjugated at an internal position of ). In some embodiments, at least one ligand is optionally conjugated via a linker or carrier to Z 11 or Z12 The ligand is conjugated to the 3' or 5' end of the oligonucleotide. In some embodiments, at least one ligand may be conjugated to a single-stranded oligonucleotide via direct linking of the oligonucleotide to a ribosugar. Alternatively, the ligand may be conjugated to a single-stranded oligonucleotide via one or more linkers (tethers) and / or carriers.

[0191] In some embodiments, the internal position is Q S This can refer to one of the 1-4 nucleotide positions upstream or downstream. In some embodiments, the internal position is Z 11 Z paired at positions 11, 12, and 13 from the 5' end 12 It can refer to one of the nucleotides located 1 to 4 nucleotides upstream or downstream of the nucleotide.

[0192] Z 12 Regarding this, for the purpose of counting the internal positions for ligand-only conjugation, Q S (Z 12 The terminal nucleotide (of which) can be considered an internal position.

[0193] In some embodiments, the internal position is from there, Q is directly connected to the loop region. S and / or Z 12 The exclusion of the nucleotides; and / or Z 11 The removal of the 2nd or 14th position from the 5' end; and / or Z 11 The 11th, 12th, and 13th positions from the 5' end are excluded; and / or Z 11 Q paired at positions 11, 12, and 13 from the 5' end S and / or Z 12 The position is excluded; and / or In equation (II) or (IIa), Z 12 The 3' end and Z 11The exclusion of two or three terminal positions from the 5' end; and / or In equation (III) or (IIIa), Z 12 The 5' end and Z 11 Two or three terminal positions are excluded from the 3' end. It can be characterized by:

[0194] In some embodiments, the ligand may be conjugated to a single-stranded oligonucleotide via a monovalent, branched divalent, or trivalent linker.

[0195] In some embodiments, the ligand may be conjugated to a single-stranded oligonucleotide via a carrier that substitutes one or more nucleotides. The carrier may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranyl, and dekalinyl. In one embodiment, the acyclic group is a portion based on a serinol skeleton or a diethanolamine skeleton.

[0196] In a particular embodiment, at least one of the ligands includes a lipophilic moiety.

[0197] In one embodiment, the lipophilic portion is a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, docosanic acid (DCA), dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, lithocholic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In a particular embodiment, the lipid is a fatty acid (omega-3 fatty acid, e.g.) selected from the group consisting of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0198] In some embodiments, the lipophilic portion is saturated or unsaturated C4-C 30 Hydrocarbon chains (for example, C4~C 30 It contains alkyl or alkenyl groups, as well as any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0199] In some embodiments, the lipophilic portion is saturated or unsaturated C6-C 18 Hydrocarbon chains (e.g., linear C6~C) 18 Alkyl or alkenyl compounds, for example, saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C) 16 Contains alkyl or alkenyl compounds. In some embodiments, the lipophilic portion is saturated or unsaturated C. 14 ~C 24 Hydrocarbon chains (e.g., linear C) 14 ~C 24 Alkyl or alkenyl compounds, for example, saturated or unsaturated C 22 Hydrocarbon chains (e.g., linear C) 22It contains alkyl or alkenyl compounds. For example, one or more non-terminal positions of a single-stranded oligonucleotide are, as described herein, "2'-C" of formula (I) [wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or a modified derivative thereof), and the n-hexadecyl chain is the lipophilic moiety]. 16 The "2'-C" modification may be present in another example. In another example, one or more non-terminal positions of a single-stranded oligonucleotide may have the "2'-C" modification of formula (2) [wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or a modified derivative thereof), and the n-docosanyl chain is the lipophilic moiety] as described herein. 22 It may have the modifier ".

[0200] n-hexadecyl chain or n-docosanyl chain, C4~C 30 Similar modifications that substitute for hydrocarbon chains include "2'-C4~C 30 This is called a "hydrocarbon chain" (or C6~C 18 Hydrocarbon chain or C 14 ~C 24 Substitution with hydrocarbon chains is "2'-C6~C 18 "Hydroxide chain" or "2'-C" 14 ~C 24 (Also known as a "hydrocarbon chain").

[0201] In related embodiments, Z 11 and Z 12 At least one or more non-terminal nucleotide positions are 2'-C4~C 30 Hydrocarbon chain structure, 2'-C6~C 18 Hydrocarbon chain structure, 2'-C 14 ~C 24 Hydrocarbon chain structure, 2'-C of formula (1) 16 Structure, or 2'-C of formula (2) 22 It has a structure.

[0202] In some embodiments, the lipophilic portion contains one or more phospholipids.

[0203] In some embodiments, the lipophilic portion comprises one or more lipids or lipophilic ligands, all of which are disclosed in International PCT Publications WO2019 / 232255A1 and WO2021 / 108662A1 and U.S. Patent No. 10,184,124, which are incorporated herein by reference in their entirety.

[0204] In some embodiments, the ligand comprises one or more ligands of formula (L-1), (L-2), (L-3), or (L-4) as described herein.

[0205] In some embodiments, ligands include those disclosed in International PCT Publications WO2017 / 053999, WO2019 / 118916, WO2022 / 031433, WO2022 / 056269, WO2022 / 056273, and WO2022 / 056277, all of which are incorporated herein by reference in their entirety.

[0206] In some embodiments, the lipophilic portion is saturated or unsaturated C4-C 30 (For example, C4~C 18 ) Contains hydrocarbon chains, as well as any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid (carboylic acid), sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0207] In some embodiments, the lipophilic portion is appropriately connected to Z via a linker or carrier. 11 or Z 12 It is conjugated to one or more internal positions.

[0208] In some embodiments, Z 11 and Z 12At least one of the nucleotide sequences includes one or more internal positions (i.e., non-terminal positions) other than positions 9–12 of the nucleotide sequence; for example, positions 4–8 and 13–18 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence as position 1; positions 5, 6, 7, 15, and 17 of the nucleotide sequence; or one or more lipophilic moieties independently conjugated to positions 4, 6, 7, and 8 of the nucleotide sequence.

[0209] In some embodiments, Z 11 and Z 12 At least one of these includes one or more lipophilic moieties independently conjugated at position 6 of the nucleotide sequence, counted from the 5' end of the nucleotide sequence. In one embodiment, Z 11 and Z 12 Each of these contains a lipophilic moiety conjugated at position 6 of the nucleotide sequence; the lipophilic moiety is saturated or unsaturated C4-C 30 (For example, C4~C 18 )Hydroxide chains, or saturated or unsaturated C 14 ~C 24 It may also contain hydrocarbon chains; the lipophilic portion may be saturated or unsaturated C 16 Hydrocarbon chains or saturated or unsaturated carbon 22 It may contain hydrocarbon chains.

[0210] In some embodiments, Z 11 and Z 12 At least one of the nucleotide sequences includes one or more non-terminal positions of the nucleotide sequence; for example, positions 6–10 and 15–18 of the nucleotide sequence, counted from the 5' end as position 1; and one or more lipophilic moieties independently conjugated at positions 15 and 17 of the nucleotide sequence.

[0211] In some embodiments, at least one lipophilic moiety is conjugated to an internal position of a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), and from the internal position, Z 11The 2nd or 14th position from the 5' end is removed; and / or Z 11 The 11th, 12th, and 13th positions from the 5' end are excluded; and / or Z 11 Q paired at positions 11, 12, and 13 from the 5' end S and / or Z 12 The position of is excluded; and / or In equation (II) or (IIa), Z 12 The 3' end and Z 11 Two or three terminal positions may be excluded from the 5' end; and / or In equation (III) or (IIIa), Z 12 The 5' end and Z 11 Two or three terminal positions may be excluded from the 3' end.

[0212] In some embodiments, at least one ligand is a targeting ligand selected from the group consisting of antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In one embodiment, at least one ligand is an integrin receptor ligand.

[0213] The targeting ligand is appropriately linked to a nucleotide sequence (e.g., Z) via a linker or carrier. 11 and Z 12 It can be conjugated at an internal position of ). Alternatively, the targeting ligand can be conjugated via a linker or carrier as appropriate, Z 11 or Z 12 It can be conjugated to the 3' or 5' end of [the specified element].

[0214] In certain embodiments, at least one of the ligands is a carbohydrate-based ligand. Carbohydrate-based ligands may be D-galactose, polyvalent galactose, N-acetyl-D-galactosamine (GalNAc), polyvalent GalNAc, D-mannose, polyvalent mannose, polyvalent lactose, N-acetyl-glucosamine, glucose, polyvalent glucose, polyvalent fucose, glycosylated polyaminoacid, or lectin.

[0215] In some embodiments, the carbohydrate-based ligand is a divalent or trivalent branched linker, for example: [ka] It is one or more GalNAc derivatives that are conjugated by Z. In some embodiments, the carbohydrate-based ligand is Z 11 Or Z 12 The 3' end, or Z 11 Or Z 12 It is conjugated to the internal position of Z 12 It is conjugated to the 3' end.

[0216] In some embodiments, one or more targeting ligands (e.g., carbohydrate-based ligands) are Z, excluding the 2nd or 14th position. 11 It is conjugated to an internal position.

[0217] In the above embodiments of the present invention relating to single-stranded oligonucleotides, the phosphate mimetic modification at the 5' end of the nucleotide sequence of the single-stranded oligonucleotide of formula (I), formula (II) (or IIa), or formula III (or IIIa) may be a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP). 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinyl phosphate), a 5'-Z-VP isomer (i.e., cis-vinyl phosphate), or a mixture thereof.

[0218] In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP). In one embodiment, the phosphate mimetic is 5'-cyclopropylphosphonate. In one embodiment, the phosphate mimetic is 5'-vinylphosphate.

[0219] In another embodiment, the single-stranded oligonucleotide is an antisense strand (i.e., Z 1 The 5' end of ) further contains a phosphate group or a phosphate mimetic. The phosphate mimetic may be a 5'-vinylphosphonate (VP). If the phosphate mimetic is a 5'-vinylphosphonate (VP), the 5' terminal nucleotide has the following structure: [ka] [In formula: X is either O or S; R is hydrogen, hydroxyl, fluoro, or C 1~20 It is an alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R 5’ The double bond between them is in the E or Z direction (for example, the E direction), and B is a nucleobase or a modified nucleobase, and B may be adenine, guanine, cytosine, thymine, or uracil. It may have.

[0220] In one embodiment, R 5’ In another embodiment, R is methoxy, and R 5’ In this embodiment, the compound is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E direction. In another embodiment, X is S, R is methoxy, and R 5’ This is =C(H)-P(O)(OH)2, with C5' carbon and R 5’ The double bond between them is in the E direction.

[0221] In some embodiments, the -CH2OH group at the 4' position of the 5' terminal nucleotide is represented by the formula -O-CH2-P(O)(OR)2[wherein each R is independently hydrogen or C]. 1~4 It is substituted with a phosphoric acid mimetic that is alkyl (for example, one R group is hydrogen and one R group is methyl; or both R groups are hydrogen).

[0222] In one embodiment, the phosphate mimetic is 5'-cyclopropylphosphonate (VP), i.e., the CH2OH group at the 4' position of the 5' terminal nucleotide is given by the formula -Cy-P(O)(OR)2[wherein Cy is a cyclopropyl ring, and each R is independently hydrogen or C]. 1~4 Substituted by alkyl groups (for example, one R group is hydrogen, or both R groups are hydrogen).

[0223] In some exemplary embodiments, the 5'-terminated phosphate mimetic is [ka] , or their salts (e.g., sodium salts), where B may be a modified nucleobase (e.g., U).

[0224] In some embodiments, the 5' terminal phosphate mimetic is part of a modified 5' terminal nucleotide. For example, the phosphate mimetic is structural [ka] [In the formula, B may be a modified nucleobase.] It may be part of a modified 5' terminal nucleotide that has [this characteristic].

[0225] In some embodiments, the 5'-terminated phosphate mimetic may also include a 5'-phosphate prodrug or a 5'-phosphonate prodrug. In some embodiments, the 5'-phosphate prodrug or 5'-phosphonate prodrug has the structure of the formula disclosed in WO2022 / 147214, which is incorporated herein by reference. In some exemplary embodiments, the 5'-phosphate prodrug or 5'-phosphonate prodrug is Pmmds( [ka] ((4SR,5SR)-3,3,5-trimethyl-1,2-dithiolan-4-ol)phosphodiester); cPmmds( [ka] ((4SR, 5RS)-3,3,5-trimethyl-1,2-dithiolan-4-ol)phosphodiester (Cis Pmmds)); PdArls( [ka] ((4SR,5RS)-5-phenyl-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester);PdAr3s( [ka] ((4SR,5RS)-5-(4-methylphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester);PdAr5s( [ka] ((4SR,5RS)-5-(4-methoxyphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol)phosphodiester);PdAr2s( [ka] );PdAr4s( [ka] );PdAr6s( [ka] );Pmmd / Pmmds( [ka] );Pmds( [ka] );Cymd / Cymds( [ka] , X is the OS); or Ptmd / Ptmds( [ka] X is the OS), Pd / Pds( [ka] (X is the OS) That is the case.

[0226] In some exemplary embodiments, a 5'-phosphate prodrug or a 5'-phosphonate prodrug is [ka] Therefore, an siRNA containing one of the above list of 5'-modified phosphate prodrugs generally has activity comparable to that of an siRNA containing 5'-VP. In some exemplary embodiments, a 5'-phosphate prodrug or a 5'-phosphonate prodrug is [ka] siRNAs containing one of the above list of 5'-modified phosphate prodrugs generally have improved stability and better or comparable activity to siRNAs containing 5'-VP.

[0227] Another aspect of the present invention relates to an oligonucleotide construct comprising two single-stranded oligonucleotides of formula (I) above, wherein the two single-stranded oligonucleotides are covalently bonded.

[0228] Another aspect of the present invention relates to an oligonucleotide construct comprising two single-stranded oligonucleotides of the above formula (II) (or IIa) or (III) (or IIIa), wherein the two single-stranded oligonucleotides are covalently bonded.

[0229] In some embodiments, at least one single-stranded oligonucleotide forming the oligonucleotide construct is derived from formula (I). In some embodiments, at least one single-stranded oligonucleotide forming the oligonucleotide construct is derived from formula (II) (or IIa). In some embodiments, at least one single-stranded oligonucleotide forming the oligonucleotide construct is derived from formula (III) (or IIIa).

[0230] In some embodiments, the covalent bond between the two single-stranded oligonucleotides is located at the linking group L of each single-stranded oligonucleotide.

[0231] In some embodiments, the two single-chain oligonucleotides are oximes, aminooxys, triazoles or fusion triazoles, phosphodiesters, phosphotriesters, hydrogen phosphonates, alkyl or aryl phosphonates, phosphoramidates, phosphorothioates, nitrogen-modified phosphorus-containing linkages (PN linkages), methylene methyliminos, thiodiesters, thionocarbamates, N,N'-dimethylhydrazines, phosphoroselenates, boranophosphates, boranophosphate esters, amides, hydroxylaminos, siloxy Covalently bonded by a tethering group selected from the group consisting of ethanol, dialkylsiloxane, carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal, formacetal, methylene imino, methylene carbonylamino, methylene hydrazo, methylene dimethylhydrazo, methylene oxymethyl imino, ether, thioacetamide, and combinations thereof.

[0232] In some embodiments, the tethering group is an oxime, aminooxy, or triazole or fusion triazole. An exemplary process for covalently bonding an exemplary tethering group and two single-stranded oligonucleotides to form an oligonucleotide construct is shown in Schemes 7.1-7.4 below.

[0233] The two single-stranded oligonucleotides may be the same or different.

[0234] In some embodiments, the two single-stranded oligonucleotides forming the oligonucleotide construct are identical. In one embodiment, the single-stranded oligonucleotides forming the oligonucleotide construct are derived from formula (I). In one embodiment, the single-stranded oligonucleotides forming the oligonucleotide construct are derived from formula (II) (or IIa). In one embodiment, the single-stranded oligonucleotides forming the oligonucleotide construct are derived from formula (III) (or IIIa).

[0235] In some embodiments, the two single-stranded oligonucleotides that form the oligonucleotide construct are different.

[0236] In some embodiments, the two single-stranded oligonucleotides forming the oligonucleotide construct are two different single-stranded oligonucleotides derived from formula (I). In some embodiments, one single-stranded oligonucleotide is Z 1 and / or Z 2 This is the Z of another single-stranded oligonucleotide. 1 and / or Z 2 It contains modifications different from those of the other single-stranded oligonucleotides. In some embodiments, L of one single-stranded oligonucleotide is different from L of the other single-stranded oligonucleotides. In some embodiments, Q of one single-stranded oligonucleotide 1 and / or Q 2 Q is the same as other single-stranded oligonucleotides. 1 and / or Q 2 This is different. In some embodiments, one single-stranded oligonucleotide contains a different ligand than the other single-stranded oligonucleotides. For example, one single-stranded oligonucleotide contains a ligand, while the other single-stranded oligonucleotides do not contain a ligand or contain a different ligand. In some embodiments, one single-stranded oligonucleotide contains a ligand located at a different position than the ligand on the other single-stranded oligonucleotides.

[0237] In some embodiments, the two single-stranded oligonucleotides forming the oligonucleotide construct are two different single-stranded oligonucleotides derived from formula (II) (or IIa) or (III) (or IIIa). In some embodiments, one single-stranded oligonucleotide is derived from formula (II) (or IIa) and the other single-stranded oligonucleotide is derived from formula (III) (or IIIa). In some embodiments, one single-stranded oligonucleotide is derived from formula Z 11 and / or Z 12 This is the Z of other single-stranded oligonucleotides. 11 and / or Z 12 It contains a different modification. In some embodiments, L of one single-stranded oligonucleotide is different from L of another single-stranded oligonucleotide. For example, one single-stranded oligonucleotide contains L, while the other single-stranded oligonucleotide does not contain L or contains a different L. In some embodiments, Q of one single-stranded oligonucleotide S Q is the same as other single-stranded oligonucleotides. S This is different. For example, one single-stranded oligonucleotide is Q S It contains Q, and the other single-stranded oligonucleotides are Q S Does not contain or has a different Q S It contains. In some embodiments, one single-stranded oligonucleotide contains a different ligand than the other single-stranded oligonucleotides. For example, one single-stranded oligonucleotide contains a ligand, while the other single-stranded oligonucleotides do not contain a ligand or contain a different ligand. In some embodiments, one single-stranded oligonucleotide contains a ligand located at a different position on the other single-stranded oligonucleotides.

[0238] In some embodiments, the two single-stranded oligonucleotides forming the oligonucleotide construct are two distinct single-stranded oligonucleotides, one derived from formula (I) and the other derived from formula (II) (or IIa) or formula (III) (or IIIa).

[0239] Another aspect of the present invention relates to a pharmaceutical composition comprising the single-stranded oligonucleotide according to formula (I) and a pharmaceutically acceptable excipient.

[0240] Another aspect of the present invention relates to a pharmaceutical composition comprising the single-stranded oligonucleotide according to formula (II) (or (IIa)) or formula (III) (or (IIIa)), and a pharmaceutically acceptable excipient.

[0241] Another aspect of the present invention relates to a pharmaceutical composition comprising the above-mentioned oligonucleotide construct, which comprises two single-stranded oligonucleotides according to formula (I), (II) (or (IIa)), or formula (III) (or (IIIa)), and a pharmaceutically acceptable excipient.

[0242] All of the above embodiments relating to single-stranded oligonucleotides, including nucleotide sequences(or more), formula (I) and all variables defined in formula (I), formula (II)-(IIa) and all variables defined in formula (II)-(IIa), formula (III)-(IIIa) and all variables defined in formula (III)-(IIIa), chemical modifications on the nucleotide sequence, linking group L within the oligonucleotide, tethering group covalently linking two single-stranded oligonucleotides, and ligands and ligand conjugations disclosed in the above embodiments of the present invention relating to single-stranded oligonucleotides are suitable for this embodiment of the present invention relating to pharmaceutical compositions.

[0243] Another aspect of the present invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising the steps of contacting the cells with the single-stranded oligonucleotide of formula (I) in an amount sufficient to inhibit the activity or expression of one or more target genes in the cells of the subject, or administering them to the subject.

[0244] Another aspect of the present invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising the steps of contacting the subject cells with the single-stranded oligonucleotide of formula (II) (or (IIa)) or (III) (or (IIIa)) in an amount sufficient to inhibit the activity or expression of one or more target genes in the subject cells, or administering them to the subject.

[0245] Another aspect of the present invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising the steps of contacting the cells with an oligonucleotide construct comprising two single-stranded oligonucleotides according to formula (I), (II) (or (IIa)) or (III) (or (IIIa)) in an amount sufficient to inhibit the activity or expression of one or more target genes in the cells of the subject, or administering the oligonucleotide construct to the subject.

[0246] All of the above embodiments relating to single-stranded oligonucleotides, including nucleotide sequences(or more), formula (I) and all variables defined in formula (I), formula (II)-(IIa) and all variables defined in formula (II)-(IIa), formula (III)-(IIIa) and all variables defined in formula (III)-(IIIa), chemical modifications on nucleotide sequences, linking groups L within oligonucleotides, tethering groups covalently linking two single-stranded oligonucleotides, and ligands and ligand conjugations disclosed in the above embodiments of the present invention relating to single-stranded oligonucleotides are suitable for this aspect of the present invention relating to methods for inhibiting the expression of one or more target genes in a subject.

[0247] In some embodiments, cells are within the scope. In one embodiment, the scope is human. In one embodiment, the scope is a non-human mammal, such as a rhesus monkey, cynomolgus monkey, mouse, or rat.

[0248] In all of the above embodiments of the present invention, a single-stranded oligonucleotide can inhibit the activity or expression of one or more target genes in the tissue of a subject by at least 15% compared to a suitable control (e.g., compared to an untreated or placebo-treated subject, or compared to a reference value, e.g., including target mRNA or protein levels in the treated subject measured before treatment with a single-stranded oligonucleotide or double-stranded nucleic acid agent), and may inhibit the activity or expression of one or more target genes in the tissue of a subject by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% compared to a suitable control. In one embodiment, the suitable control is an untreated subject. In one embodiment, the suitable control is a reference value, e.g., a value obtained in the subject before administration of a single-stranded oligonucleotide to the subject. [Brief explanation of the drawing]

[0249]

Figure 1A

Figure 1B

[0250]

Figure 2

[0251]

Figure 3

[0252]

Figure 4

[0253]

Figure 5A

Figure 5B

Figure 5C

[0254]

Figure 6

[0255]

Figure 7

[0256]

Figure 8A

Figure 8B

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Figure 9

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Figure 10

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Figure 11A

Figure 11B

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Figure - 12

[0261] The inventors have designed a novel strategy for preparing single-stranded loop oligonucleotides using two chemically modified oligonucleotides capable of forming an intrachain double-strand region and linking two oligonucleotides by a cleavage-type linking group to generate a single-stranded construct. The single-stranded loop oligonucleotides are designed to undergo cleavage at a suitable rate so that the single-stranded construct is cleaved into a double-stranded RNAi agent that is effective in vivo. The single-stranded loop oligonucleotides are synthesized as single-stranded, self-annealed by sequence complementarity, and purified as single-stranded. Delivery ligands such as tritactile GalNAc can be readily incorporated during synthesis. The single-stranded loop oligonucleotides described herein have stability in plasma due to their ability to effectively metabolize and release siRNA in vivo. The single-stranded loop oligonucleotides discussed herein simplify the production and purification of RNAi agents by increasing throughput and reducing overall synthesis time, while simultaneously providing an improved design so that the efficacy of the RNAi agent is preserved or improved when cleaved in vivo. Single-chain oligonucleotide structure design

[0262] One aspect of the present invention is formula (I): (5'-Z 1 -3')-Q 1 -LQ 2 -(5'-Z 2 -3') (I) [In formula: Z 1 This is a first oligonucleotide containing 15 to 100 appropriately modified nucleotides that are substantially complementary to the target gene; Z 2 is, Z 1 A second oligonucleotide containing 15 to 100 appropriately modified nucleotides, substantially complementary to the first oligonucleotide; Z 1 and Z 2 It can form an intrachain double-strand region containing three or more consecutive base pairs; L is a linking group; Q1 and Q 2 Each of these independently represents a modified nucleotide between 0 and 12. A single-stranded oligonucleotide having a sequence represented by , which can inhibit the expression of a target gene, At least one nucleotide in formula (I) is a modified nucleotide. Regarding single-stranded oligonucleotides.

[0263] Single-stranded oligonucleotides are formed by linking two oligonucleotides together with a linking group L. Some exemplary single-stranded oligonucleotide constructs are illustrated in Scheme 1 and 2. [ka] [ka]

[0264] As shown in schemes 1 and 2, in some embodiments, Z 1 represents the first oligonucleotide (e.g., antisense strand) that is substantially complementary to the target gene; Z 2 is, Z 1 A second oligonucleotide (e.g., a sense chain) that is substantially complementary to the first oligonucleotide. In some embodiments, Z 1 and Z 2 is, Z 1 and Z 2 An intrachain double-strand region can be formed between corresponding nucleotides, and a single-strand oligonucleotide contains a loop region formed by a linking group L (and possibly Q). 1 and Q 2 ). In some embodiments, Q 1 and Q 2 Each of these may not exist independently. In some embodiments, Q 1 and Q 2 Each of these independently corresponds to the first oligonucleotide Z. 1 and the second oligonucleotide Z2 It may exist as an overhang. As shown in Schemes 1 and 2, L is a linking group that may contain modified or unmodified nucleotides. In some embodiments, as shown in Scheme 2, L may contain a non-nucleotide-based linker such as Q304. In some embodiments, as shown in Schemes 1 and 2, (Z 1 , Z 2 Q 1 Q 2 The nucleotides of the entire single-stranded oligonucleotide (including L) may contain various chemical modifications such as DNA, RNA, 2'-F, or 2'-OMe. In some embodiments, as shown in schemes 1 and 2, the single-stranded oligonucleotide is Z 2 (For example, a sense chain) has a trivalent branched linker at its 3' end. [ka] The ligand conjugated by, for example, a 3GalNAc derivative, further comprises Z 1 The molecule further comprises a phosphate group or a phosphate mimetic (e.g., a 5'-terminal vinyl phosphonate (5'-VP)) at the 5' end of the antisense chain (e.g., an antisense chain).

[0265] First oligonucleotide Z 1 and the second oligonucleotide Z 2 Each of these may be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. First oligonucleotide Z 1 and the second oligonucleotide Z 2Each of these may have a length of approximately 10 to approximately 50 nucleotides, approximately 10 to approximately 40 nucleotides, approximately 10 to approximately 35 nucleotides, approximately 10 to approximately 30 nucleotides, approximately 10 to approximately 25 nucleotides, approximately 10 to approximately 20 nucleotides, approximately 15 to approximately 50 nucleotides, approximately 15 to approximately 40 nucleotides, approximately 15 to approximately 35 nucleotides, approximately 15 to approximately 30 nucleotides, approximately 15 to approximately 25 nucleotides, approximately 15 to approximately 20 nucleotides, or approximately 18 to approximately 20 nucleotides. In one embodiment, the first oligonucleotide Z 1 and the second oligonucleotide Z 2 Each of them is at least 15 nucleotides long. In one embodiment, the first oligonucleotide Z 1 and the second oligonucleotide Z 2 Each of them is at least 18 nucleotides long.

[0266] Another aspect of the present invention is, Formula (II) or (III): (5'-Z 11 -3')-LQ S -(5'-Z 12 -3') (II), (3'-Z 11 -5')-LQ S -(3'-Z 12 -5') (III), [In formula: Z 11 This is a first oligonucleotide containing 15 to 100 appropriately modified nucleotides that are substantially complementary to the target gene; Z 12 is, Z 11 A second oligonucleotide containing 10 to 100 appropriately modified nucleotides, substantially complementary to the first oligonucleotide; Z 11 and Z 12 This can form an intrachain double-strand region containing 7 or more consecutive base pairs; Q S This represents appropriately modified nucleotides from 0 to 12. L is any linking group. A single-stranded oligonucleotide by At least one nucleotide in formula (II) is a modified nucleotide; and At least one nucleotide in formula (III) is a modified nucleotide, where, In equation (II), Z 11 At least one nucleotide at the 3' end of formula (III), Z 11 At least one nucleotide at the 5' end is L and Q in both cases. S Along with Z 11 and Z 12 Forms a loop region that connects Regarding single-stranded oligonucleotides.

[0267] Single-stranded oligonucleotides are formed by connecting two oligonucleotides using appropriate linking groups L.

[0268] In some embodiments, the single-stranded oligonucleotide is defined as formula (IIa) or formula (IIIa): [ka] [In formula: Z 11 This includes W-LP, W is Z 12 This forms an intrachain double helix region of at least 7 base pairs, LP, along with L as appropriate, has W and Z at its 3' or 5' end. 12 Forming a loop between them, [ka] This represents any existence of L, [ka] Q S Represents any existence of, [ka] is, Z 11Represents any overhang at the 5' or 3' end of the object. [ka] is, Z 12 [Represents any overhang at the 5' or 3' end] It is represented by [this].

[0269] Some exemplary single-stranded oligonucleotides are illustrated in schemes 1B.1–1B.5 and schemes 2B.1–2B.4.

[0270] Some exemplary single-stranded oligonucleotides are defined by formula (II) or (IIa), as illustrated by schemes 1B.1 to 1B.5, Z 11 and Z 12 It may have a direction (e.g., 5'-3' direction) and connections. The PS nucleotide junctions illustrated in each of schemes 1B.1 to 1B.5 are illustrative and may or may not be present. In a particular embodiment, the illustrated PS nucleotide junctions are present at the 3' and 5' ends, but there are no internal PS nucleotide junctions. [ka]

[0271] As shown in Scheme 1B.1, in some embodiments, Z 11 represents the first oligonucleotide that is substantially complementary to the target gene (e.g., the antisense strand); Z 12 is, Z 11 A second oligonucleotide that is substantially complementary to (for example, the sense chain). In some embodiments, Z 11 and Z 12 is, Z 11 and Z 12 An intrachain double-strand region can be formed between corresponding nucleotides, while a single-stranded oligonucleotide contains a loop region LP (possibly containing a linking group L; not marked).

[0272] In some embodiments, Q S Q may not exist. In some embodiments, Q S It is represented by a and b, which may be a spacer, Z 11 These can be any appropriately modified nucleotides that form matched or mismatched base pairs with their opposite nucleotides (e.g., the two corresponding nucleotides at positions 17 and 18 in scheme 1B.1). In one embodiment, both a and b are Z 11 They form a match base pair with their opposite nucleotides. In one embodiment, both a and b are Z 11 It forms a mismatch base pair with the opposite nucleotide. In one embodiment, one of a and b is Z 11 The opposite nucleotide forms a matching base pair, and the other a and b form Z 11 It forms a mismatch base pair with the opposite nucleotide. In one embodiment, b is Z 11 It forms a mismatch base pair with the opposite nucleotide (for example, b is mismatched with the nucleotide at position 17, as shown in scheme 1B.1). In one embodiment, a is Z 11 It forms a mismatch base pair with the opposite nucleotide (for example, a is mismatched with the nucleotide at position 18, as shown in Scheme 1B.1).

[0273] In some embodiments, the single-stranded oligonucleotide is Z 11 or Z 12 The first 6 nucleotides or the last 6 nucleotides contain one or two phosphorothioate internucleotide linkage modifications (e.g., two consecutive phosphorothioate internucleotide linkage modifications) (i.e., Z 11 or Z 12 (One or two phosphorothioate internucleotide ligation modifications between nucleotides at terminal 6, either from the 5' or 3' end). In one embodiment, the single-stranded oligonucleotide is Z 11 or Z12 The first three nucleotides or the last three nucleotides contain two consecutive phosphorothioate nucleotide linkage modifications (i.e., Z 11 or Z 12 The internucleotide junction between either the 5' or 3' end and terminal 3 is modified by two consecutive phosphorothioate internucleotide junction modifications, e.g., the phosphorothioate internucleotide junction modification shown as a "star" in scheme 1B.1 iii).

[0274] In some embodiments, the single-stranded oligonucleotide is Z 11 The last 8 nucleotides contain one or two phosphorothioate internucleotide linkage modifications (e.g., two consecutive phosphorothioate internucleotide linkage modifications). In one embodiment, the single-stranded oligonucleotide is 23 nucleotides long Z 11 It has two phosphorothioate internucleotide ligation modifications between the nucleotides at positions 16-23 (for example, as shown in scheme 1B.1, Z 11 It contains two phosphorothioate nucleotide linkage modifications between the nucleotides at positions 16-17, 17-18, 18-19, 19-20, 20-21, and 21-22.

[0275] In some embodiments, the single-stranded oligonucleotide is Z 12 The first three nucleotides contain one or two phosphorothioate internucleotide ligation modifications (e.g., two consecutive phosphorothioate internucleotide ligation modifications) (e.g., as shown in Scheme 1B.1, Z 12 (One or two phosphorothioate internucleotide ligation modifications between the nucleotides at positions 1-2 and / or 2-3). In one embodiment, the single-stranded oligonucleotide is Z 12 Two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3, Z 12Two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides (e.g., positions 14-15 and 15-16), and as shown in scheme 1B.1 ii), Z 11 It contains two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3.

[0276] In some embodiments, as shown in scheme 1B.1 ii), the single-stranded oligonucleotide has a nucleotide sequence (e.g., Z 11 and / or Z 12 At the 5' end of ), as shown in the 5'-phosphate modification or 5'-phosphate mimetic modification described herein (e.g., scheme 1B.1 ii), Z 11 It may contain a 5'-terminal vinyl phosphonate (5'-VP) at its 5' end.

[0277] In some embodiments, the single-stranded oligonucleotide may further comprise one or more ligands (e.g., lipophilic moieties for extrahepatic delivery as described herein). In some embodiments, one or more lipophilic moieties may be Z 11 It is independently conjugated to one or more internal positions (i.e., non-terminal positions) of the . In one embodiment, one or more lipophilic portions are Z as shown in ii) of Scheme 1B.1 11 The 5' end is independently conjugated to one or more positions 11, 12, and 13. In one embodiment, one or more lipophilic moieties are Z, excluding position 2 or 14. 11 It is independently conjugated to one or more internal positions of the . In one embodiment, one or more lipophilic portions are Z as shown in scheme 1B.1 ii). 11 Q, which is positioned at the 5' end, is paired with the 11th, 12th, and 13th positions. S and / or Z 12 Excluding the position of Z 12 and / or Q S It is independently conjugated to one or more positions.

[0278] In some embodiments, the single-stranded oligonucleotide may further comprise one or more targeting ligands (e.g., liver-targeting carbohydrate-based ligands as described herein). In some embodiments, one or more targeting ligands (e.g., carbohydrate-based ligands) may be (as shown in iii) of Scheme 1B.1) 11 Or Z 12 The 3' end, or Z 11 Or Z 12 It is conjugated to an internal position. In some embodiments, one or more targeting ligands (e.g., carbohydrate-based ligands) are conjugated to positions Z, excluding positions 2 or 14. 11 It is conjugated to an internal position. In some embodiments, one or more targeting ligands (e.g., carbohydrate-based ligands) are conjugated to Z as shown in iii) of Scheme 1B.1. 12 It is conjugated to the 3' end.

[0279] In some embodiments, if a single-stranded oligonucleotide contains a ligand terminal conjugation to the 5' or 3' terminal nucleotide, or contains a debasalized nucleotide, inverted nucleotide, or inverted debasalized nucleotide terminal conjugation to the 5' or 3' terminal nucleotide, then the above-mentioned internucleotide linkage modification to the terminal nucleotide may be removed at its end (for example, as shown in iii of Scheme 1B.1), Z 12 Conjugation of the ligand to the 3' end of Z 12 (One or two phosphorothioate nucleotide ligation modifications between the terminal 6th or 3rd nucleotides may be removed from the 3' end.) [ka]

[0280] In some embodiments, as shown in scheme 1B.2, Z 11 It contains 19-23 appropriately modified nucleotides, Z12 It contains 12-16 appropriately modified nucleotides, Q S It contains two appropriately modified nucleotides.

[0281] In some embodiments, as shown in Scheme 1B.2, non-loop ends (e.g., Z 11 Z at the 5' end 11 and Z 12 The double-stranded region formed by this process has a blunt end. [ka]

[0282] In some embodiments, as shown in scheme 1B.3, Z 11 It contains 19-23 appropriately modified nucleotides, Z 12 It contains 12-16 appropriately modified nucleotides, Q S It contains two appropriately modified nucleotides.

[0283] In some embodiments, as shown in Scheme 1B.3, L, Q S , or Z 12 Connected, Z 11 The 3 to 5 terminal nucleotides contain modifications selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-ara nucleotide (aN) to ensure cleavage. In some embodiments, L, Q S , or Z 12 Connected, Z 11 The five terminal nucleotides are, #-dN-dN-fN-fN-fN- ** , #-dN-dN-rN-dN-dN- ** , #-dN-dN-rN-rN-rN- ** , #-dN-dN-dN-dN-dN- ** , #-mN-mN-fN-fN-fN- ** , #-mN-mN-dN-dN-dN- ** , #-mN-mN-rN-dN-dN- ** , #-mN-mN-rN-rN-rN- ** , and [In formula: # is Z 11 It is a coupling to, ** L, Q S , or Z 12 It is a coupling to dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents a ribonucleotide, and mN represents 2'-O-methylnucleotide. It has modifications selected from the group consisting of the following.

[0284] In some embodiments, as shown in Scheme 1B.3, L, Q S , or Z 12 Connected, Z 11 The three terminal nucleotides are 2'-fluoro, 2'-deoxy, and 2'-OH, for example. #-fN-fN-fN- ** , #-dN-dN-dN- ** , #-dN-dN-rN- ** , #-dN-rN-dN- ** , #-rN-dN-dN- ** , and #-rN-rN-dN- ** , #-rN-dN-rN- ** , #-dN-rN-rN- ** , #-rN-rN-rN- ** It has modifications that are independently selected from the group consisting of the following.

[0285] In one embodiment, as shown in scheme 1B.3, the single-stranded oligonucleotide is Z 11 Two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3, and Z 12 It contains two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides.

[0286] In one embodiment, as shown in scheme 1B.3, the single-stranded oligonucleotide is modified with six terminal phosphorothioate nucleotide linkage modifications; Z 11 Two consecutive phosphorothioate nucleotide ligation modifications between nucleotides at positions 1-2 and 2-3, Z 12 Two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides, and Z 12 It contains two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3.

[0287] In some embodiments, as shown in Scheme 1B.3, the second oligonucleotide Z 12 Q as appropriate S It also contains at least one motif of three consecutive 2'-F modifications, the nucleotide following the motif is not 2'-F modified. In some embodiments, the positions of the three consecutive modification motifs are as follows: The motif is Q S , Z 12 They are in 1st and 2nd place, Z 11 It may be 19 nucleotides long; The motif is Z 12 It is in 1st, 2nd, and 3rd place, Z 11 It may be 20 nucleotides long; The motif is Z 12 They are in 2nd, 3rd and 4th place, Z 11 It may be 21 nucleotides long; The motif is Z 12 It is in 3rd, 4th and 5th place, Z 11It may be 22 nucleotides long; or The motif is Z 12 It is in 4th, 5th and 6th place, Z 11 It may be 23 nucleotides long; It is characterized by one of the following.

[0288] In some embodiments, as shown in Scheme 1B.3, the first oligonucleotide Z 11 It contains modifications other than 2'-O-methyl at the 2nd and 14th positions. In one embodiment, as shown in scheme 1B.3, the first oligonucleotide Z 11 It contains a 2'-F modification at position 14.

[0289] In some embodiments, as shown in Scheme 1B.3, the first oligonucleotide Z 11 It contains one or more 2'-deoxy(DNA) modifications at positions 2, 5, 7, and 12.

[0290] In some embodiments, as shown in scheme 1B.3, Z 11 and Z 12 All remaining modifications are 2'-O-methyl modifications. [ka]

[0291] In some embodiments, as shown in scheme 1B.4, Z 11 It contains 19-23 appropriately modified nucleotides, Z 12 It contains 12-16 appropriately modified nucleotides, Q S It contains two appropriately modified nucleotides.

[0292] In some embodiments, as shown in Scheme 1B.4, L, Q S , or Z 12 Connected, Z 11The 3 to 5 terminal nucleotides contain modifications selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-ara nucleotide (aN) to ensure cleavage. In some embodiments, L, Q S , or Z 12 Connected, Z 11 The five terminal nucleotides are, #-dN-dN-fN-fN-fN- ** , #-dN-dN-rN-dN-dN- ** , #-dN-dN-rN-rN-rN- ** , #-dN-dN-dN-dN-dN- ** , #-mN-mN-fN-fN-fN- ** , #-mN-mN-dN-dN-dN- ** , #-mN-mN-rN-dN-dN- ** , #-mN-mN-rN-rN-rN- ** , and [In formula: # is Z 11 It is a coupling to, ** L, Q S , or Z 12 It is a coupling to dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents ribonucleotide, mN represents 2'-O-methylnucleotide. It has modifications selected from the group consisting of the following.

[0293] In some embodiments, as shown in Scheme 1B.4, L, Q S , or Z 12 Connected, Z 11The three terminal nucleotides are 2'-fluoro, 2'-deoxy, and 2'-OH, for example. #-fN-fN-fN- ** , #-dN-dN-dN- ** , #-dN-dN-rN- ** , #-dN-rN-dN- ** , #-rN-dN-dN- ** , #-rN-rN-dN- ** , #-rN-dN-rN- ** , #-dN-rN-rN- ** , and #-rN-rN-rN- ** It has modifications that are independently selected from the group consisting of the following.

[0294] In one embodiment, as shown in scheme 1B.4, the single-stranded oligonucleotide is Z 11 Two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3, and Z 12 It contains two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides.

[0295] In one embodiment, as shown in Scheme 1B.4, the single-stranded oligonucleotide is modified with six terminal phosphorothioate nucleotide linkage modifications; Z 11 Two consecutive phosphorothioate nucleotide ligation modifications between nucleotides at positions 1-2 and 2-3, Z 12 Two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides, and Z 12 It contains two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3.

[0296] In some embodiments, as shown in Scheme 1B.4, a second oligonucleotide Z 12Q as appropriate S It also contains at least one motif of three consecutive 2'-F modifications, the nucleotide following the motif is not 2'-F modified. In some embodiments, the positions of the three consecutive modification motifs are as follows: The motif is Q S , Z 12 They are in 1st and 2nd place, Z 11 It may be 19 nucleotides long; The motif is Z 12 It is in 1st, 2nd, and 3rd place, Z 11 It may be 20 nucleotides long; The motif is Z 12 They are in 2nd, 3rd and 4th place, Z 11 It may be 21 nucleotides long; The motif is Z 12 It is in 3rd, 4th and 5th place, Z 11 It may be 22 nucleotides long; or The motif is Z 12 It is in 4th, 5th and 6th place, Z 11 It may be 23 nucleotides long. It is characterized by one of the following.

[0297] In some embodiments, as shown in scheme 1B.4, Z 12 Q as appropriate S Along with that, its position is Z 11 Unless it is part of the motif, it contains a 2'-O-methyl or 2'-F modification at a position two positions prior to the motif of three consecutive 2'-F modifications (n-2 position if the motif starts at position n). In one embodiment, as shown in scheme 1B.4, Z 12 Q as appropriate S Along with that, its position is Z 11 Unless it is part of a motif, it contains a 2'-F modification at a position two positions before a motif of three consecutive 2'-F modifications (position n-2 if the motif starts at position n).

[0298] In some embodiments, as shown in Scheme 1B.4, the first oligonucleotide Z 11It contains modifications other than 2'-O-methyl at the 2 and 14 positions. In one embodiment, as shown in scheme 1B.4, the first oligonucleotide Z 11 It contains a 2'-F modification at position 14.

[0299] In some embodiments, as shown in Scheme 1B.4, the first oligonucleotide Z 11 It contains one or more 2'-F modifications at positions 2, 6, 8, 9, 14, and 16.

[0300] In some embodiments, as shown in scheme 1B.4, Z 11 and Z 12 All remaining modifications are 2'-O-methyl modifications. [ka]

[0301] In some embodiments, L exists in formula (II) (or IIa) or formula III (or IIIa), and formula:#-(N) n - ** It contains a connecting part represented by . In this formula, # is Z 11 It is a coupling to, ** Q S or Z 12 It is a bond to; n is 3 to 12; and each N is independently a linked monomer having a chain length of 3 or more atoms. In some embodiments, n is 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5. In one embodiment, n is 3.

[0302] In some embodiments, as shown in Scheme 1B.5, all linked monomers of L (e.g., Q304) together with LP, W(Z 11 ) and Z 12 A loop is formed between them. In some embodiments, as shown in scheme 1B.5, one or more linked monomers of L (e.g., Q304) together with LP, W(Z 11 ) and Z 12A loop is formed between them, and one or more linked monomers of L (e.g., Q304) are not in the loop region. In some embodiments, as shown in scheme 1B.5, one or more linked monomers of L (e.g., Q304) together with LP, W(Z 11 ) and Z 12 A loop is formed between them, and one or more linked monomers of L (e.g., Q304) are not in the loop, Q S (a) is connected to LP. In some embodiments, as shown in scheme 1B.5, one or more linked monomers of L (e.g., Q304) are connected to W(Z) together with LP. 11 ) and Z 12 A loop is formed between them, and one or more linked monomers of L (e.g., Q304) are not in the loop, Z 12 It connects to the network.

[0303] In some embodiments, one or more linkages (N) in L may be appropriately modified nucleotides. In some embodiments, one or more linkages (N) in L may be independently selected from the group consisting of 2'-deoxynucleotides (dN), 2'-deoxy-2'-fluoronucleotides (fN), ribonucleotides (rN), 2'-O-methylnucleotides (mN), and 2'-ara nucleotides (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide).

[0304] In some embodiments, one or more connecting portions (N) in L may be independently selected from the group consisting of Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, and Q368.

[0305] In some embodiments, as shown in Scheme 1B.5, L contains a triplet of Q304.

[0306] In some embodiments, as shown in Scheme 1B.4, the single-stranded oligonucleotide is Z 11 Two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3, Z 12 Two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides, and Z 12 It contains two consecutive phosphorothioate nucleotide ligation modifications between the nucleotides at positions 1-2 and 2-3.

[0307] In some embodiments, as shown in Scheme 1B.5, the single-stranded oligonucleotide is Z 11 The last 5, 6, or 7 nucleotides contain one or two phosphorothioate internucleotide ligation modifications (e.g., two consecutive phosphorothioate internucleotide ligation modifications).

[0308] In one embodiment, as shown in scheme 1B.5, the single-stranded oligonucleotide is modified with six terminal phosphorothioate nucleotide linkage modifications; Z 11 Two consecutive phosphorothioate nucleotide ligation modifications between nucleotides at positions 1-2 and 2-3, Z 12 Two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides, and Z 12 It contains two consecutive phosphorothioate nucleotide linkage modifications between nucleotides at positions 1-2 and 2-3.

[0309] In one embodiment, as shown in Scheme 1B.5, the single-stranded oligonucleotide is modified with eight terminal phosphorothioate nucleotide linkage modifications; Z 11 Two consecutive phosphorothioate internucleotide ligation modifications between nucleotides at positions 1-2 and 2-3; Z 12 A ligation modification between two consecutive phosphorothioate nucleotides between the last three nucleotides; Z 12 Two consecutive phosphorothioate internucleotide ligation modifications between nucleotides at positions 1-2 and 2-3; and Z 11The last 5, 6, or 7 nucleotides contain two consecutive phosphorothioate nucleotide ligation modifications.

[0310] Some exemplary single-stranded oligonucleotides are defined by formula (III) or (IIIa), as illustrated by schemes 2B.1 to 2B.4, Z 11 and Z 12 It may have a direction (e.g., 5'-3' direction) and connections. [ka] [ka] [ka] [ka]

[0311] In some embodiments, Z 11 It contains 19-23 appropriately modified nucleotides, Z 12 It contains 16-19 appropriately modified nucleotides, Q S It may or may not be present, or it may be present containing two appropriately modified nucleotides.

[0312] In some embodiments, as shown in scheme 2B.1, Z 11 It contains 23 appropriately modified nucleotides, Z 12 It contains 16-19 appropriately modified nucleotides. In some embodiments, as shown in scheme 2B.2, Z 11 It contains 21 appropriately modified nucleotides, Z 12 It contains 14-17 appropriately modified nucleotides. In some embodiments, as shown in scheme 2B.3, Z 11 It contains 23 appropriately modified nucleotides, Z 12It contains 18-21 appropriately modified nucleotides. In some embodiments, as shown in scheme 2B.4, Z 11 It contains 21 appropriately modified nucleotides, Z 12 It contains 16 to 19 appropriately modified nucleotides.

[0313] In some embodiments, as shown in schemes 2B.3 and 2B.4, non-loop ends (e.g., Z 11 Z at the 3' end 11 and Z 12 The double-stranded region formed by this process has a blunt end.

[0314] In some embodiments, Z at the non-loop end 11 It has an overhang of 1 to 3 nucleotides in length. In one embodiment, Z at the non-loop end 11 It has an overhang of 2 nucleotides (for example, as shown in schemes 2B.1 and 2B.2, Z 11 (3' end). In one embodiment, as shown in schemes 2B.1 and 2B.2, Z at the non-loop end. 11 It has an overhang of 2 nucleotides in length, and a phosphorothioate nucleotide linkage between the two overhanging nucleotides.

[0315] In one embodiment, as shown in schemes 2B.1 and 2B.2, Z at the non-loop end 11 It has an overhang of 2 nucleotides in length (for example, Z 11 (at the 3' end), it has two phosphorothioate nucleotide links between the three terminal nucleotides (for example, Z 11 At the 3' end, two of the three nucleotides are overhanging nucleotides, and the third is the next pairing nucleotide of the overhanging nucleotide.

[0316] In some embodiments, as shown in schemes 2B.1 and 2B.2, the single-stranded oligonucleotide is Z 11 Within the first 4 nucleotides or Z12 The first three nucleotides contain one or two phosphorothioate internucleotide ligation modifications (e.g., two consecutive phosphorothioate internucleotide ligation modifications).

[0317] In some embodiments, as shown in schemes 2B.1 to 2B.4, the single-stranded oligonucleotide is modified with six terminal phosphorothioate nucleotide linkage modifications; Z 12 Two consecutive phosphorothioate internucleotide ligation modifications between nucleotides at positions 1-2 and 2-3; Z 11 Two consecutive phosphorothioate internucleotide ligation modifications between the first four nucleotides; and Z 11 It contains two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides.

[0318] In some embodiments, as shown in schemes 2B.1 to 2B.4, the single-stranded oligonucleotide is modified with eight terminal phosphorothioate nucleotide linkage modifications; Z 12 Two consecutive phosphorothioate internucleotide ligation modifications between nucleotides at positions 1-2 and 2-3; Z 12 Intercellular linkage modification between two consecutive phosphorothioate nucleotides between the last three nucleotides; Z 11 Two consecutive phosphorothioate nucleotide ligation modifications between the first four nucleotides; and Z 11 It contains two consecutive phosphorothioate nucleotide ligation modifications between the last three nucleotides.

[0319] In some embodiments, if a single-stranded oligonucleotide contains a ligand terminal conjugation to the 5' or 3' terminal nucleotide, or contains a debasalized nucleotide, an inverted nucleotide, or an inverted debasalized nucleotide terminal conjugation to the 5' or 3' terminal nucleotide, then the above-mentioned internucleotide linkage modification to the terminal nucleotide may be removed at its end (for example, as shown in schemes 2B.1 to 2B.4, Z 12Conjugation of the ligand to the 5' end of Z 12 (One or two phosphorothioate internucleotide ligation modifications between nucleotides from the terminal to position 6 or position 3 at the 5' end may be removed.)

[0320] In some embodiments, as shown in schemes 2B.1 to 2B.4, Z 11 and Z 12 The intrachain double-strand region formed by may contain all consecutive base pairs or up to 3 (e.g., 0, 1, 2, or 3) mismatched base pairs. In one embodiment, Z 12 is, Z 11 It may contain one nucleotide that forms a mismatch base pair with the opposite nucleotide (for example, Z 12 The last nucleotide, or the (n-1)th nucleotide if the last nucleotide is the nth nucleotide.

[0321] In some embodiments, as shown in schemes 2B.1 to 2B.4, L, Q S , or Z 12 Connected, Z 11 The 3 to 5 terminal nucleotides contain modifications selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-ara nucleotide (aN) to ensure cleavage. In some embodiments, L, Q S , or Z 12 Connected, Z 11 The five terminal nucleotides are, #-dN-dN-fN-fN-fN- ** , #-dN-dN-rN-dN-dN- ** , #-dN-dN-rN-rN-rN- ** , #-dN-dN-dN-dN-dN- ** , #-mN-mN-fN-fN-fN- ** , #-mN-mN-dN-dN-dN- ** 、 #-mN-mN-rN-dN-dN- ** 、and #-mN-mN-rN-rN-rN- ** 、 has a modification selected from the group consisting of.

[0322] [[ID=十七]]In some embodiments, as shown in Schemes 2B.1 to 2B.4, L, Q S , or Z 12 [[ID=2十一]]The three terminal nucleotides of Z connected to 11 [[ID=二十三]]are 2'-fluoro, 2'-deoxy, and 2'-OH, for example #-fN-fN-fN- ** 、 #-dN-dN-dN- ** 、 #-dN-dN-rN- ** [[ID=3十五]]、 #-dN-rN-dN- ** 、 #-rN-dN-dN- ** 、 #-rN-rN-dN- ** 、 #-rN-dN-rN- ** 、 #-dN-rN-rN- ** 、and #-rN-rN-rN- ** has a modification independently selected from the group consisting of.

[0323] In some embodiments, L is present in Formula (II) (or IIa) or Formula III (or IIIa), and the formula: #-(N) n - ** contains a linking moiety represented by. In this formula, # is the bond to Z 11 is the bond to, ** [[ID=七十二]]is Q S or Z 12It is a bond to; n is 3 to 12; and each N is independently a linked monomer having a chain length of 3 or more atoms. In some embodiments, n is 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5. In one embodiment, n is 3.

[0324] In some embodiments, as shown in schemes 2B.1 to 2B.4, all linked monomers of L (e.g., Q304) together with LP, W(Z 11 ) and Z 12 A loop is formed between them. In some embodiments, as shown in schemes 2B.1 to 2B.4, one or more linked monomers of L (e.g., Q304) together with LP, W(Z 11 ) and Z 12 A loop is formed between them, and one or more linked monomers of L (e.g., Q304) are not in the loop region. In some embodiments, as shown in schemes 2B.1 to 2B.4, one or more linked monomers of L (e.g., Q304) together with LP, W(Z 11 ) and Z 12 A loop is formed between them, and one or more linked monomers of L (e.g., Q304) are not in the loop, Q S (a) is connected to LP. In some embodiments, as shown in schemes 2B.1 to 2B.4, one or more linked monomers of L (e.g., Q304) are connected to W(Z) together with LP. 11 ) and Z 12 A loop is formed between them, and one or more linked monomers of L (e.g., Q304) are not in the loop, Z 12 It connects to the network.

[0325] In some embodiments, one or more linkages (N) in L may be appropriately modified nucleotides. In some embodiments, one or more linkages (N) in L may be independently selected from the group consisting of 2'-deoxynucleotides (dN), 2'-deoxy-2'-fluoronucleotides (fN), ribonucleotides (rN), 2'-O-methylnucleotides (mN), and 2'-ara nucleotides (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide).

[0326] In some embodiments, one or more connecting portions (N) in L may be independently selected from the group consisting of Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, and Q368.

[0327] In one embodiment, as shown in schemes 2B.1 to 2B.4, L contains a triplet of Q304.

[0328] Single-stranded oligonucleotide sequences can be substrates that can be cleaved by DICER.

[0329] Another aspect of the present invention relates to an oligonucleotide construct comprising two single-stranded oligonucleotides of formula (I), as described above, wherein the two single-stranded oligonucleotides are covalently bonded. In some embodiments, the two single-stranded oligonucleotides are covalently bonded via a tethering group. Exemplary tethering groups and exemplary processes for covalently bonding two single-stranded oligonucleotides to form an oligonucleotide construct are shown in the following schemes 7.1 to 7.4.

[0330] A particular embodiment of the present invention relates to a linking group design for connecting two oligonucleotides to form a single-stranded oligonucleotide. A particular embodiment of the present invention relates to a tethering group design (i.e., bivalent chromosome style) for connecting two oligonucleotides to form an oligonucleotide construct. Linker / Tether

[0331] Linkers / tethers are contained within the linking group L of a single-stranded oligonucleotide and can connect two oligonucleotides to form a single-stranded oligonucleotide.

[0332] Linkers / tethers are contained within the tethering group of an oligonucleotide construct (i.e., in a bivalent chromosome style) and can link two single-stranded oligonucleotides to form an oligonucleotide construct.

[0333] Linkers / tethers can also be used, for example, to attach ligands to single-stranded oligonucleotides via a support.

[0334] The terms "linker," "linking," "linking base," "linking portion," and "tether" may be used interchangeably.

[0335] The linking group L may contain multiple linkers / tethers, each of which may be the same or different.

[0336] The linking group L of a single-stranded oligonucleotide can be a nucleotide-based or non-nucleotide-based linker. Linking group L can be a stable linker that is stable in body fluids (e.g., plasma or artificial cerebrospinal fluid). Alternatively, linking group L can be a cleavage-type linker (e.g., a biological cleavage-type linker).

[0337] Linkers / tethers can be connected to ligands at the "tethering junction (TAP)". Linkers / tethers can be connected to any C1-C 100 Carbon-containing portion (for example, C1~C 75 , C1~C50 , C1~C 20 , C1~C 10 ;C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 ) may contain at least one nitrogen atom. In certain embodiments, the nitrogen atom may form a portion of the terminal amino or amide (NHC(O)-) group of the linker / tether and be used as a ligand connection point. Non-limiting examples of linkers / tethers (underlined) include TAP- (CH 2 ) n ;TAP- NH- 2 ) n ;TAP- C(O)(CH 2 ) n , TAP- NH- 2 ) n ;TAP- NR’’’’(CH 2 ) n ;TAP- NH- ;TAP- 2 ) n C(O)-(CH ;TAP- 2 ) n - ;TAP- -C(O)- ;TAP- ;TAP- C(O)-(CH 2 ) n ;TAP- -C(O)O- 2 ) n ;TAP- C(O)-O- 2 ) n - ; or TAP- 2 ) n [[ID=Z8]]C(O)-(CHThe linker / tether may be substituted with, for example, hydroxy, alkoxy, or perhaloalkyl groups, and / or may be inserted with one or more further heteroatoms, such as N, O, or S. Preferred tethered ligands are, for example, TAP- 2 ) n -NH-C(O)- ;TAP- 2 ) n C(O)-(CH ;TAP- 2 ) n C(O)-NH- ;TAP- 2 ) n C(O)- ;TAP- 2 ) n (CH ;TAP- 2 ) n -C(O)- ;TAP- 2 ) n (CH 2 ,TAP- -C(O)O- 2 ) n 2 (CH ;TAP- 2 ) n (CH 2 ;TAP- [[ID=Z9]]-NH-C(O)- 2 ) n ;TAP- (CH 2 ) n ;TAP- NH(LIGAND) ;TAP- 2 ) n [[ID=Z2]]C(O)(CH ;TAP- 2 ) n NH(LIGAND) ;TAP- ;TAP- NR’’’’ (CH ;TAP- 2 ) n NH(LIGAND) ;TAP- 2 ) n (CH ;TAP- 2 ) n ONH(LIGAND) ; or TAP- 2 ) n C(O)(CH This may include: In some embodiments, the amino-terminal linker / tether (e.g., NH2, ONH2, NH2NH2) may form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminal linker / tether (e.g., NH2, ONH2, NH2NH2) may be acylated by, for example, C(O)CF3.

[0338] In some embodiments, the linker / tether may be terminated by a mercapto group (i.e., SH) or an olefin (e.g., CH=CH2). For example, the tether may be TAP- 2 ) n ONH(LIGAND) , TAP- 2 ) n NR’’’’(CH , TAP- 2 ) n ONH(LIGAND) 2 ) , or TAP- 2 ) n (CH 2 )It may also be as follows, where n may be as described elsewhere in this specification. The tether may be optionally substituted with, for example, hydroxy, alkoxy, or perhaloalkyl, and / or may be optionally inserted with one or more further heteroatoms, such as N, O, or S. The double bond may be cis or trans, or E or Z.

[0339] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at the terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, aldehydes, alkyl halides, mesylates, tosylates, nosylates, or brosylates, or activated carboxylic acid esters, such as NHS esters or pentafluorophenyl esters. A preferred linker / tether (underlined) is TAP- 2 ) n NHNH ;TAP- 2 ) n (LIGAND) ; or TAP- 2 ) n C(O)(CH (where n is 1-6 and R'''' is a C1-C6 alkyl); or TAP- 2 ) n NHNH ;TAP- 2 ) n (LIGAND) ; or TAP- 2 ) n NR’’’’(CH (Here, n is 1 to 6, and R'''' is a C1 to C6 alkyl group); TAP- 2 ) n NHNH 6 F 5; TAP- 2 ) n (LIGAND) 6 F 5; or TAP- 2 )n C(O)-(CH 6 F 5 (where n is from 1 to 11 and R'''' is C1 - C6 alkyl); or 2 ) n -C(O)(LIGAND) 2 ; TAP - C(O)-(CH 2 ) n 2 -C(O)O(LIGAND) ; or TAP - 2 ) n C(O)-O(LIGAND) 2 C(O)-(CH -NH-C(O)(LIGAND) C(O)-(CH (LIGAND) C(O)-NH(LIGAND) C(O)(LIGAND) (CH -C(O)(LIGAND) (CH -C(O)O(LIGAND) (CH (LIGAND) (CH -NH-C(O)(LIGAND) (CH -SH C(O)(CH SH (CH -(CH=CH C(O)(CH (CH=CH (CH CHO C(O)(CH CHO NR’’’’(CH CHO (CH C(O)ONHS C(O)(CH C(O)ONHS NR’’’’(CH C(O)ONHS (CH C(O)OC C(O)(CH C(O)OC NR’’’’(CH C(O)OC -(CH CH LG C(O)(CH CH LG NR’’’’(CH CH LG Note: In the original text, there seems to be an error in line 16 where it says "

Figure - 12

Figure 12

[0340] 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

[0341] [[ID=第41]] In other embodiments, other protected amino groups, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (where the aryl moiety can be ortho - nitrophenyl, or ortho, para - dinitrophenyl) can be at the terminal position of the linker / tether.

[0342] Any linker / tether described herein can further include one or more additional linking groups, such as -O-(CH2) n -, -(CH2) n -SS -, -(CH2) n -, or -(CH = CH)-.

[0343] Cutting-type linker / tether In some embodiments, at least one linker / tether may be a redox-cleaving linker, an acid-cleaving linker, an esterase-cleaving linker, a phosphatase-cleaving linker, a peptidase-cleaving linker, or an endosome-cleaving linker.

[0344] In one embodiment, at least one linker / tether may be a reductive cleavage type linker (e.g., a disulfide group).

[0345] In one embodiment, at least one linker / tether may be an acid-cleaving linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group).

[0346] In one embodiment, at least one linker / tether may be an esterase-cleaving linker (e.g., an ester group).

[0347] In one embodiment, at least one linker / tether may be a phosphatase-cleaving linker (e.g., a phosphate group).

[0348] In one embodiment, at least one linker / tether may be a peptidase-cleaving linker (e.g., a peptide bond).

[0349] In one embodiment, at least one linker / tether may be an endosomal cleavage linker (or a protease cleavage linker, e.g., a carbohydrate linker). For example, a carbohydrate linker is cleaved at least 1.25 times 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).

[0350] Severable linkers are sensitive to the presence of severing agents, such as pH, redox activity, or degradable molecules. Generally, severing agents are found more frequently, at high levels, or with high activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selected for a particular substrate or do not have substrate specificity, such as oxidases or reductases, or reducing agents such as mercaptans that are present in cells and can degrade redox-severable linkers by reduction; esterases; endosomes, or factors that can create an acidic environment, such as an acidic environment resulting in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-severable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0351] Scleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is somewhat lower, ranging from approximately 7.1 to 7.3. Endosomes have a highly acidic pH in the range of 5.5 to 6.0, and lysosomes have a still highly acidic pH of approximately 5.0. Some tethers may be cleaved at a favorable pH, thereby possessing linking groups that release iRNA agents from intracellular ligands (e.g., targeting or cell-permeable ligands, e.g., cholesterol) or into desired cellular compartments.

[0352] The chemical linkage (e.g., linking group) that connects the ligand to the iRNA agent may include a disulfide bond. When the iRNA agent / ligand complex is taken up into a cell by endocytosis, the acidic environment of the endosome causes the disulfide bond to break, 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 may be a secondary therapeutic agent that can complement the therapeutic effect of the targeting ligand or iRNA agent.

[0353] A tether may contain a linking group that is cleaved by a specific enzyme. The type of linking group incorporated into the tether may depend on the cell being targeted by the iRNA agent. For example, an iRNA agent that targets mRNA in liver cells may be conjugated to a tether containing an ester group. Since liver cells are rich in esterases, the tether will likely be cleaved more efficiently in liver cells than in esterase-unriched cell types. Cleavage of the tether may release the iRNA agent from the ligand conjugated to the distal end of the tether, thereby enhancing the silencing activity of the iRNA agent. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

[0354] A tether containing a peptide bond can be conjugated to an iRNA agent and targeted to peptidase-rich cell types, such as liver cells and synovial cells. For example, an iRNA agent that targets synovial cells, such as for the treatment of inflammatory diseases (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond.

[0355] Generally, the suitability of a candidate cleavage ligator can be evaluated by testing the ability of a degrading agent (or degradation conditions) to cleave the candidate ligator. It may also be desirable to investigate the candidate cleavage ligator's resistance to cleavage in the blood or in contact with other non-target tissues, such as tissues to which the iRNA agent is exposed when administered to a subject. Therefore, the relative sensitivity to cleavage between the first and second conditions can be determined, where the first condition is selected to indicate cleavage within target cells, and the second condition is selected to indicate cleavage in other tissues or in body fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ cultures or tissue cultures, or in whole animals. It may be useful to perform initial evaluations under cell-free or culture conditions and then confirm them with further evaluations in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at a rate of at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0356] The cleavage linker may be capable of cleaving various tissue and cellular structures, such as homogenates, tritosomes, cytosols, or endosomes of any type of cell, for example, liver homogenates, liver tritosomes, liver lysosomes, liver cytosols, liver endosomes, brain homogenates, brain tritosomes, brain lysosomes, brain cytosols, or brain endosomes.

[0357] Redox-cut type connector One class of cleavage-type linkers is the redox-cleavage-type linker, which is cleaved upon reduction or oxidation. An example of a reductive-cleavage-type linker is the disulfide linker (-SS-). Methods described herein can be considered to determine whether a candidate cleavage-type linker is a suitable “reductive-cleavage-type linker” or whether it is suitable for use with, for example, a specific iRNA moiety and a specific targeting agent. For example, a candidate substance may be evaluated by incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate observed intracellularly, for example, in a target cell. The candidate substance may also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved to a maximum of 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at a rate of at least 2, 4, 10, or 100 times in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound may be determined using a standard enzyme kinetic assay under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0358] Phosphate-based cleavage-type linking groups Phosphate-based linking groups are cleaved by factors that degrade or hydrolyze the phosphate group. In cells, examples of factors that cleave the phosphate group include enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidate linkages can be evaluated using methods similar to those described above.

[0359] Acid-cleavable linking group Acid-cleaved linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments, acid-cleaved linking groups are cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by factors such as enzymes that can act as general acids. Within cells, specific low-pH organelles, such as endosomes and lysosomes, provide a cleavage environment for acid-cleaved linking groups. Examples of acid-cleaved linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and amino acid esters. Acid-cleaved groups may have the general formula -C=NN-, C(O)O, or -OC(O). Preferred embodiments are those in which 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 candidate linking groups can be evaluated using methods similar to those described above.

[0360] Ester-based linking groups Ester-based linking groups are cleaved by enzymes such as esterases and amidases within cells. Examples of ester-based cleaving linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleaving linking groups have the general formulas C(O)O- or -OC(O)-. These candidate linking groups can be evaluated using methods similar to those described above.

[0361] Peptide-based cleavage groups Peptide-based linkers are cleaved by enzymes such as peptidases and proteases within cells. Peptide-based cleavage linkers are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavage linkers do not contain amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. Peptide bonds are a special type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleavage linkers are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and do not include the entire amide functional group. The general formula for a peptide cleavage linker is -NHCHR 1 C(O)NHCHR 2 C(O)-[wherein, R 1 and 2 [These have the R groups of two adjacent amino acids.] These candidates can be evaluated using methods similar to those described above. Biological cutting linker / tether

[0362] Linkers may also include biocleavage linkers, which are nucleotide and non-nucleotide linkers or combinations thereof that connect two parts of a molecule. For example, a biocleavage linker may be used as part of a linking group L that connects two oligonucleotides of a single-stranded oligonucleotide. In some embodiments, a simple electrostatic or stacking interaction between two individual nucleotide sequences may represent a linker.

[0363] Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and their derivatives, as well as aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0364] In some embodiments, at least one linker (tether) is a biological cleavage linker selected from the group consisting of DNA, RNA, disulfides, amides, galactosamine functional monosaccharides or oligosaccharides, glucosamine, glucose, galactose, and mannose, as well as combinations thereof.

[0365] In some embodiments, the cleavage linker (or biocleavage linker) contains one or more carbohydrate (sugar) moieties and / or peptide linkers. The cleavage linker (or biocleavage linker) may be used to link two nucleotide sequences or oligonucleotides, to link a nucleotide sequence or oligonucleotide to a ligand, or to link a ligand to an endosome cleavage agent.

[0366] In some embodiments, the bio-cleaving carbohydrate linker has the following characteristics: i) Biological cleavage type carbohydrate linkers may have 1 to 10 sugar units. ii) The sugar portion has at least one anomeric linkage that can connect two nucleotide sequences or oligonucleotides. iii) If two or more sugars are present, these nucleotide sequences or oligonucleotides may be linked via 1-3, 1-4, or 1-6 sugar linkages. iv) If two or more sugars are present, these nucleotide sequences or oligonucleotides may also be linked via alkyl chains. It has one or more of these.

[0367] An example of a biological cleavage linker is, [ka] JPEG2026516488000076.jpg240169 JPEG2026516488000077.jpg239169 JPEG2026516488000078.jpg218167[In the formula, n=1~12 and m=1~12] Includes.

[0368] In some embodiments, the cleavage linker (or biocleavage linker) belongs to the following group: [ka] JPEG2026516488000080.jpg177167 An endosomal cleavage linker comprising one or more sugar units independently selected from the given group.

[0369] In some embodiments, the endosomal linker comprises two or more of the above-mentioned sugar units.

[0370] In some embodiments, the endosomal linker contains 1 to 10 sugar units.

[0371] In some embodiments, the endosomal linker contains 2 to 10 sugar units. In some embodiments, the sugar units of the endosomal linker are selected from the group consisting of Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, and Q317.

[0372] In some embodiments, the endosomal cleavage linker comprises two, three, or four saccharide units. In some embodiments, the saccharide unit is selected from the group consisting of Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, and Q317. For example, the saccharide unit may be Q304.

[0373] In one embodiment, an endosomal cleavage linker is provided. -Q303Q303-, -Q303Q303Q303-, -Q303Q303Q303Q303-, -Q304Q304-, -Q304Q304Q304-, -Q304Q304Q304Q304-, -Q305Q305-, -Q305Q305Q305-, -Q306Q306-, -Q306Q306Q306-, -Q312Q312-, -Q312Q312Q312-, -Q313Q313-, -Q313Q313Q313-, -Q314Q314-, -Q314Q314Q314-, -Q315Q315-, -Q315Q315Q315-, -Q316Q316-, -Q316Q316Q316-, -Q317Q317-, or -Q317Q317Q317- Includes.

[0374] In one embodiment, the endosomal cleavage linker is [ka] It also includes.

[0375] In one embodiment, the endosomal cleavage linker is -Q198Q48Q303Q303Q48-, -Q198Q303Q48Q303-, -Q198Q48Q303Q303Q48-, -Q198Q303Q48Q303-, -Q198Q303Q303Q303Q303-, -Q198Q303Q303Q303-, -Q198Q303Q303-, -Q198Q304Q304Q304Q304-, -Q198Q304Q304Q304-, -Q198Q304Q304-, -Q198Q48Q303Q303Q48-, -Q198Q303Q48Q303-, -Q198Q303Q303-, -Q48Q303Q303Q48-, or -Q303Q48Q303- Includes.

[0376] Further discussion of biological linkers can be found in WO2018136620, the full content of which is incorporated herein by reference. Carrier

[0377] In certain embodiments, the linking group L connecting two single-stranded oligonucleotides contains one or more carriers. In certain embodiments, one or more ligands are conjugated to the single-stranded oligonucleotide via one or more carriers. In some embodiments, the carriers may be substituted with one or more nucleotides.

[0378] The support may be a cyclic or acyclic group. In one embodiment, 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, pyridadinyl, tetrahydrofuryl, and dekalinyl. In one embodiment, the acyclic group is a portion based on a serinol skeleton or a diethanolamine skeleton.

[0379] In some embodiments, the support is a single-stranded oligonucleotide (e.g., Z 1 and / or Z 2 Replace one or more nucleotides at internal positions(s) of the nucleotide sequence of ).

[0380] In this specification, a ribonucleotide subunit in which the ribose sugar of the subunit is replaced in this manner is referred to as a ribose substitution-modified subunit (RRMS). The carrier may be cyclic or acyclic and may include two "skeletal junctions" (e.g., hydroxyl groups) and a ligand. The ligand may be directly linked to the carrier or indirectly linked to the carrier by an intervening linker / tether, as described above. [ka]

[0381] The ligand-conjugate monomer subunit is a single-stranded oligonucleotide with a nucleotide sequence (e.g., Z 1 and / or Z 2 The ligand-conjugate monomer subunit may be the 5' or 3' terminal subunit of the oligonucleotide, that is, 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 a single-stranded oligonucleotide.

[0382] Sugar substitution-based monomers, e.g., ligand-conjugate monomers (cyclic type) Cyclic sugar-substituted monomers, such as sugar-substituted ligand-conjugate monomers, are also referred to herein as RRMS monomer compounds. The support may have the general formula (LCM-2) provided below (in its structure, the preferred skeletal junction is R 1 Or R 2 , R 3 Or R 4 , or Y is CR 9 R 10 If R 9 and R 10 (The two positions can be selected from the two skeletal junctions, e.g., R) 1 and R 4 , or R 4 and R 9 The preferred tethering junction is R 7 ;If X is CH2, then R 5 or R 6 This includes. The carrier is described below as an entity that can be incorporated into the chain. Thus, the structure has one (in the case of terminal positions) or two (in the case of internal positions) junctions, for example, R 1 Or R 2 , R 3 or 4 , or R 9 Or R10 (Y is CR 9 R 10 In this case, it is understood that this also includes situations where the R group is connected to a phosphate group or a modified phosphate group, for example, a sulfur atom containing a skeleton. For example, one of the R groups named above may be -CH2-, where one bond is connected to a support and the other to a skeleton atom, for example, oxygen or a central phosphorus atom. [ka] [In formula: X is N(CO)R 7 , NR 7 or CH2; Y is NR 8 O, S, CR 9 R 10 and; Z is CR 11 R 12 It is either true or does not exist; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of these are OR a and / or (CH2) n Ure b If so, each R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 H, OR a and / or (CH2) n Ure b And; Each R 5 , R 6 , R 11 , and R 12 These are, independently, ligand, H, and 1-3R. 13 C1-C6 alkyl or C(O)NHR which may be substituted by 7 is; or R 5 and R 11 Both are R 14A C3-C8 cycloalkyl group which may be substituted by; R 7 R may be a ligand, for example, 7 R d Is it okay, or R 7 For example, the tethering part, for example, NR c R d C1~C substituted by 20 Alkyl, or NHC(O)R d C1~C substituted by 20 The ligand may also be indirectly tethered to the carrier via an alkyl group; R 8 is H or C1-C6 alkyl; R 13 These are hydroxyl, C1-C4 alkoxy, or halo; R 14 , NR C R 7 and; R 15 is a C1-C6 alkyl or C2-C6 alkenyl which may be substituted with cyano; R 16 C1~C 10 It is alkyl; R 17 It is a liquid-phase or solid-phase supported reagent; L is -C(O)(CH2) q C(O)-, or -C(O)(CH2) q It is S-; R a This refers to a protecting group, for example, CAr3 (e.g., dimethoxytrityl group) or Si(X 5 ')(X 5 '')(X 5 ''') and here, (X 5 '), (X 5 ''), and (X 5 ''') is described elsewhere in this specification, R b P(O)(O-)H, P(OR 15 )N(R 16 )2 or LR 17and; R C is H or C1-C6 alkyl; R d is H or a ligand; Each Ar may be independently substituted with a C1-C4 alkoxy, or C6-C4 alkoxy. 10 It is Ariel, n is between 1 and 4; q is between 0 and 4.

[0383] An example of a carrier is, for example, X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 And Z does not exist; or X is N(CO)R 7 or NR 7 And Y is CR 9 R 10 And Z is CR 11 R 12 Either X is N(CO)R 7 or NR 7 And Y is O and Z is CR 11 R 12 Either X is CH2 and Y is CR 9 R 10 And Z is CR 11 R 12 And R 5 and R 11 They together form a C6 cycloalkyl (H, z=2) or an 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 This includes those that together form a C5 cycloalkyl (H, z=1) molecule.

[0384] In certain embodiments, the carrier is 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 9 R 10This can be based on the fact that Z does not exist (D). [ka] OFG 1 Preferably, it is bonded to one of the carbon atoms of the five-membered ring, to the first carbon, for example, an extra-ring alkylene group, for example, a methylene group (D's CH2OFG 1 ). OFG 2 Preferably, it is directly bonded to one of the five-membered ring carbons (D OFG 2 ). In pyrroline-based carriers, CH2OFG 1 It may be joined to C-2, OFG 2 It may be joined to C-3; or -CH2OFG 1 It may be joined to C-3, OFG 2 It may be joined to C-4. In a particular embodiment, CH2OFG 1 and OFG 2 This may be pair-substituted with one of the carbons referenced above. In 3-hydroxyproline-based supports, -CH2OFG 1 It may be joined to C-2, OFG 2 It may be joined to C-4. Pyrroline- and 4-hydroxyproline-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, which are limited by the presence of a ring. Thus, CH2OFG 1 and OFG 2 The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may arise as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2The centers having the configuration may both have the R configuration; or both may 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. A preferred example of the carrier D is as follows: [ka] Includes.

[0385] In certain embodiments, the support 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 That is the case. [ka] OFG 1 Preferably, it is bonded to one of the carbon atoms of the six-membered ring, to the first carbon, for example, an extra-ring alkylene group, for example, a methylene group (n=1) or an ethylene group (n=2) (E-(CH2) n OFG 1 ). OFG 2 Preferably, it is directly bonded to one of the carbon atoms of the six-membered ring (E-OFG 2 ) -(CH2) n OFG 1 and OFG 2 The groups may be arranged in a paired manner on 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 They may be arranged adjacent to the ring, that is, both groups may be bonded to adjacent ring carbon atoms, for example, -(CH2) n OFG 1 It may be joined to C-2, OFG 2 It may also be joined to C-3;-(CH2) n OFG 1It may be joined to C-3, OFG 2 It may also be joined to C-2;-(CH2) n OFG 1 It may be joined to C-3, OFG 2 It may be joined to C-4; or -(CH2) n OFG 1 It may be joined to C-4, OFG 2 It may be joined to C-3. Piperidine-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, which are limited by the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may arise as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having the configuration may both have the R configuration; or both may 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.

[0386] In certain embodiments, the support may be based on a piperidine ring system (F), for example, X is N(CO)R 7 or NR 7 And Y is NR 8 And Z is CR 11 R 12 Alternatively, it may be based on a morpholine ring system (G), for example, X is N(CO)R 7 or NR 7 Y is O, and Z is CR 11 R 12 That is the case. [ka] OFG 1 Preferably, it is bonded to one of the carbon atoms of the six-membered ring, to the first carbon, for example, an extra-ring alkylene group, for example, a methylene group (F or G -CH2OFG). 1 ). OFG 2 Preferably, it is directly bonded to one of the carbon atoms of the six-membered ring (OFG of F or G). 2 ). In both F and G, -CH2OFG 1 It may be joined to C-2, OFG 2 It may be joined to C-3; or vice versa. In a particular embodiment, CH2OFG 1 and OFG 2 This may be pair-substituted with one of the carbons referenced above. Piperazine- and morpholine-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, resulting from the presence of a ring. Thus, CH2OFG 1 and OFG 2 The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may arise as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having the configuration may both have the R configuration; or both may 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 at both F and G.

[0387] In certain embodiments, the support may be based on a decalin ring system, for example, where X is CH2 and Y is CR 9 R 10 And Z is CR 11 R 12 And R5 and R 11 Both may form a C6 cycloalkyl (H, z=2) or be based on an indane ring system, for example, if X is CH2 and Y is CR 9 R 10 And Z is CR 11 R 12 And R 5 and R 11 Both form a C5 cycloalkyl (H, z=1) structure. [ka] OFG 1 Preferably, the first carbon atom is bonded to one of C-2, C-3, C-4, or C-5, for example, an extra-ring methylene group (n=1) or an ethylene group (n=2) [H-(CH2) n OFG 1 OFG 2 Preferably, it is directly bonded to one of C-2, C-3, C-4, or C-5 (H-OFG 2 ) -CH2OFG 1 and OFG 2 The groups may be arranged in a paired manner on the ring, that is, both groups may be bonded to the same carbon, for example, C-2, C-3, C-4, or C-5. Alternatively, -(CH2) n OFG 1 and OFG 2 They may be arranged adjacent to the ring, that is, both groups may be bonded to adjacent ring carbon atoms, for example, -(CH2) n OFG 1 It may be joined to C-2, OFG 2 It may also be joined to C-3;-(CH2) n OFG 1 It may be joined to C-3, OFG 2 It may also be joined to C-2;-(CH2) n OFG 1 It may be joined to C-3, OFG 2 It may be joined to C-4; or -(CH2) n OFG 1It may be joined to C-4, OFG 2 It may also be joined to C-3;-(CH2) n OFG 1 It may be joined to C-4, OFG 2 It may be joined to C-5; or -(CH2) n OFG 1 It may be joined to C-5, OFG 2 It may be joined to C-4. Decalin or indan-based monomers may therefore contain bond rotations that are restricted by certain linkages, such as carbon-carbon bonds, which are limited by the presence of a ring. Thus, -(CH2) n OFG 1 and OFG 2 The isomers can be cis or trans with respect to each other in any of the pairs described above. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may arise as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having C-1 and C-6 may both have the R configuration; or both may have the S configuration; or one center may have the R configuration and the other center may have the S configuration, and vice versa. In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to each other. The tethering junction is preferably C-6 or C-7.

[0388] Other carriers may include those based on 3-hydroxyproline (J). [ka] Therefore, -(CH2) n OFG 1 and OFG 2These can be cis or trans with respect to each other. Therefore, all cis / trans isomers are clearly included. Monomers may contain one or more chiral centers and therefore may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomer mixtures. All such isomeric forms of monomers are clearly included (e.g., CH2OFG 1 and OFG 2 The centers having the configuration may both have the R configuration; or both may 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.

[0389] Further details regarding more representative cyclic, sugar-substituted-based carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0390] Sugar-substituted monomers (acyclic) Acyclic sugar-substituted monomers, such as sugar-substituted ligand-conjugate monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers are those of formula LCM-3 or LCM-4: [ka] It may have.

[0391] In some embodiments, each x, y, and z can be independently 0, 1, 2, or 3. In formula LCM-3, if y and z are different, then the third carbon can have either an R configuration or an S configuration. In preferred embodiments, x is zero, and y and z are each 1 in formula LCM-3 (e.g., based on selinol), and y and z are each 1 in formula LCM-3. Each of the following formulas LCM-3 or LCM-4 may be substituted with, for example, hydroxy, alkoxy, or perhaloalkyl.

[0392] More details on more representative acyclic, sugar-substituted base carriers can be found in U.S. Patents 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

[0393] In some embodiments, the single-stranded oligonucleotide is a nucleotide sequence (e.g., Z 1 and / or Z 2 It contains one or more ligands conjugated to the 5' end of ).

[0394] In a particular embodiment, the ligand is transmitted via a carrier and / or linker to a nucleotide sequence (e.g., Z 1 and / or Z 2 It is conjugated to the 5' end of ). In one embodiment, the ligand is of formula: [ka] A nucleotide sequence (e.g., Z) is transmitted via a carrier. 1 and / or Z 2 It is conjugated to the 5' end of ). R is the ligand.

[0395] In some embodiments, the single-stranded oligonucleotide is a nucleotide sequence (e.g., Z 1 and / or Z 2 It contains one or more ligands conjugated to the 3' end of ).

[0396] In a particular embodiment, the ligand transmits a nucleotide sequence (e.g., Z) via a carrier and / or linker. 1 and / or Z 2 It is conjugated to the 3' end of ). In one embodiment, the ligand is of formula: [ka] A nucleotide sequence (e.g., Z) is transmitted via a carrier. 1 and / or Z 2It is conjugated to the 3' end of ). R is the ligand.

[0397] In some embodiments, the ligand is linked to a nucleotide sequence (e.g., Z) via one or more linkers (tethers) and / or carriers. 1 and / or Z 2 ) is conjugated to a nucleotide sequence (e.g., Z) via one or more linkers (tethers). In one embodiment, the ligand is conjugated to a nucleotide sequence (e.g., Z) via one or more linkers (tethers). 1 and / or Z 2 It is conjugated to ).

[0398] In one embodiment, the ligand is transmitted via a cyclic carrier, and optionally via one or more interposing linkers (tethers), through a nucleotide sequence (e.g., Z 1 and / or Z 2 It is conjugated to the 5' or 3' end of ).

[0399] In some embodiments, the ligand is at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ) are conjugated to one or more internal positions on the sequence. An internal position in a nucleotide sequence refers to any nucleotide at any position in the nucleotide sequence, excluding terminal positions from the 3' and 5' ends (for example, two positions: excluding position 1 when counting from the 3' end and position 1 when counting from the 5' end).

[0400] In one embodiment, the ligand is at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ) is conjugated to one or more internal positions on and includes all positions from each end of the nucleotide sequence except for the two terminal positions (e.g., four positions: excluding positions 1 and 2 when counting from the 3' end and positions 1 and 2 when counting from the 5' end). In one embodiment, the lipophilic portion is at least one nucleotide sequence (e.g., Z 1 and / or Z 2) Conjugated to one or more internal positions on the sequence, including all positions from each end of the nucleotide sequence except for the three terminal positions (e.g., 6 positions: excluding positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).

[0401] In one embodiment, the ligand is at least one nucleotide sequence (e.g., Z) excluding the cleavage site region of the nucleotide sequence. 1 and / or Z 2 ) is conjugated at one or more internal positions on the nucleotide sequence, for example, the ligand is not conjugated at positions 9-12 counting from the 5' end of the nucleotide sequence, for example, the ligand is conjugated at one or more internal positions on the nucleotide sequence (e.g., Z 1 and / or Z 2 ) is not conjugated at positions 9-11 when counting from the 5' end. Alternatively, the internal position is the nucleotide sequence (e.g., Z 1 and / or Z 2 ) Excluding positions 11-13 when counting from the 3' end. In one embodiment, the internal positions exclude positions 12-14 when counting from the 5' end of the nucleotide sequence.

[0402] In one embodiment, the ligand is at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ) Conjugated at one or more internal positions on the nucleotide sequence, counting from the 3' end to positions 11-13 of the nucleotide sequence, and counting from the 5' end to the nucleotide sequence (e.g., Z 1 and / or Z 2 Excluding positions 12-14.

[0403] In one embodiment, one or more ligands are conjugated to one or more of the following internal positions: counting from the 5' end, the nucleotide sequence (e.g., Z 1 and / or Z 2 ) positions 4-8 and 13-18, as well as the nucleotide sequence (e.g., Z 1 and / or Z2 ) 6th to 10th place and 15th to 18th place.

[0404] In one embodiment, one or more ligands are conjugated to one or more of the following internal positions: counting from the 5' end, the nucleotide sequence (e.g., Z 1 and / or Z 2 ) positions 5, 6, 7, 15, and 17, as well as the nucleotide sequence (e.g., Z 1 and / or Z 2 ) ranked 15th and 17th.

[0405] In some embodiments, the ligand is conjugated to a nucleobase, sugar moiety, or nucleoside linkage of a single-stranded oligonucleotide. Ligand

[0406] In certain embodiments, a single-stranded oligonucleotide is further modified by the covalent joining of one or more conjugate groups. Generally, conjugate groups modify one or more properties of the joined single-stranded oligonucleotide, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, intracellular distribution, uptake into cells, charge, and clearance. Conjugate groups are commonly used in the chemical art and are joined directly to parent compounds such as oligomeric compounds, or via any linking moiety or linking group. A preferred list of conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0407] In some embodiments, the single-stranded oligonucleotide further comprises a targeting ligand that targets a receptor that mediates delivery to a specific CNS tissue. These targeting ligands may be conjugated in combination with a lipophilic moiety to enable specific local (e.g., intrathecal) and systemic delivery.

[0408] Exemplary targeting ligands that target receptor-mediated delivery to CNS tissues include peptide ligands such as Angiopep-2, lipoprotein receptor-associated protein (LRP) ligands, bEnd.3 cell-binding ligand; transferrin receptor (TfR) ligands (which can utilize the iron transport system in the brain and cargo transport to the brain parenchyma); manose receptor ligands (which target olfactory nerve sheath cells and glial cells), glucose transporter proteins, and LDL receptor ligands.

[0409] In some embodiments, the single-stranded oligonucleotide further comprises a targeting ligand that targets a receptor that mediates delivery to specific ocular tissues. These targeting ligands may be conjugated in combination with a lipophilic moiety to enable specific local (e.g., intrathecal) and systemic delivery. Exemplary targeting ligands that target receptor-mediated delivery to ocular tissues include lipophilic ligands such as all-trans retinol (targeting retinoic acid receptors); RGD peptides such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO: 1) or cyclo(-Arg-Gly-Asp-D-Phe-Cys) (SEQ ID NO: 2) (targeting retinal pigment epithelial cells); LDL receptor ligands; and carbohydrate-based ligands (targeting posterior ocular endothelial cells).

[0410] Preferred conjugate groups suitable for the present invention include lipid moieties such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids 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, e.g., 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. Acids Res., 1990, 18, 3777); polyamine chains or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine moiety or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.(Includes , 1996, 277, 923).

[0411] In general, various entities, such as ligands, can be coupled to the oligomeric compounds described herein. Ligands may include naturally occurring molecules, recombinant molecules, or synthetic molecules. Examples of ligands include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphatidine, polyethyleneimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide mimetic polyamine, dendrimer polyamine, arginine, amidine, protoamine, cationic lipids, cationic porphyrin, quaternary salts of polyamines, tyrofoam Pins, melanotropins, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyasparates, aptamers, asialofetin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, glucose-albumin conjugates, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C) , porphyrins (e.g., TPPC4, texaphyllin, saffrin), heterocyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules [e.g., steroids, bile acids, cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric 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], peptides (e.g., alpha-helix peptides, amphiphilic peptides, RGD peptides, cell-permeable peptides, endosomal lysating / membrane fusion peptides), alkylating agents, phosphates, amino acids, mercaptos, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complex of tetraaza macrorings), dinitrophenyl, HRP, AP, antibodies, form This includes, but is not limited to, hormone receptors, lectins, carbohydrates, polyunsaturated carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, p38MAP kinase activators, NF-κB activators, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlaquinolide, latruncrine A, phalloidin, swinholide A, indanosine, myoservin, tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell permeabilizers (e.g., helix-type cell permeabilizers).

[0412] Peptides and peptide mimetic ligands include naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides in which one or more amide-i.e., peptide bonds are substituted with one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules capable of folding into defined three-dimensional structures, similar to natural peptides. Peptides or peptide mimetic ligands may be about 5 to 50 amino acid lengths, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths.

[0413] Exemplary amphiphilic peptides include, but are not limited to, secropine, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidine, ceratotoxin, S. clava peptide, hyacinthenial antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaceptin, melittin, pleurosidine, H2A peptide, Xenopus peptide, esculentinis-1, and caerin.

[0414] As used herein, the term “endosomal lysis ligand” refers to a molecule having endosomal lysis properties. Endosomal lysis ligands facilitate the lysis of the composition of the present invention or its components, and / or their transport from cellular compartments such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular endoplasmic reticulum to the cytoplasm. Some exemplary endosome-lysing ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and branched polyamines, e.g., spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo- or polycation or anion, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers having masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptide mimetic compounds, pH-sensitive peptides, natural and synthetic membrane-fusion lipids, and natural and synthetic cationic lipids.

[0415] Exemplary endosomal lysis / membrane fusion peptides include: AALEAAEALEALALEALAEAAAAGGC(GALA)(SEQ ID NO: 3); AALEALAEALAEALAEALAEALAAAAGGC(EALA)(SEQ ID NO: 4); ALEALAEALEALAEA(SEQ ID NO: 5); GLFEAIEGFIENGWEGMIWDYG(INF-7)(SEQ ID NO: 6); GLFGAIAGFIENGWEGMIDGWYG(Inf HA-2)(Sequence ID 7);GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMIDGWYGC(diINF-7)(Sequence ID 8);GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC(diINF-3)(Sequence ID 9);GLFGALAEALAEALAEHLAEALAEALEALAAGGSC(GLF)(Sequence ID 10);GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC(GALA-INF3)(Sequence ID 11);GLFEAIEGFIENGWEGnIDGKGLFEAIEGFIENGWEGnIDG(INF-5,n is no This includes, but is not limited to, leulocine (SEQ ID NO: 12); LFEALLELLESLWELLLEA(JTS-1)(SEQ ID NO: 13); GLFKALLKLLKSLWKLLLKA(ppTG1)(SEQ ID NO: 14); GLFRALLRLLRSLWRLLLRA(ppTG20)(SEQ ID NO: 15); WEAKLAKALAKALAKHLAKALAKALKACEA(KALA)(SEQ ID NO: 16); GLFFEAIAEFIEGGWEGLIEGC(HA)(SEQ ID NO: 17); GIGAVLKVLTTGLPALISWIKRKRQQ(Melittin)(SEQ ID NO: 18); H5WYG(SEQ ID NO: 19); and CHK6HC(SEQ ID NO: 20).

[0416] To put it simply, without being bound by theory, membrane fusion lipids fuse with the membrane, consequently destabilizing it. Membrane fusion lipids typically have a small head group and an unsaturated acyl chain. Exemplary membrane-fusion lipids include, but are not limited to, 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloylphosphatidylcholine (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)methaneamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethaneamine (also referred to herein as XTC).

[0417] Synthetic polymers having endosomal lysis activity suitable for the present invention are described in U.S. Patent Publications 2009 / 0048410, 2009 / 0023890, 2008 / 0287630, 2008 / 0287628, 2008 / 0281044, 2008 / 0281041, 2008 / 0269450, 2007 / 0105804, 20070036865, and 2004 / 0198687, the contents of which are incorporated herein by reference in their entirety.

[0418] Examples of cell-permeable peptides include RQIKIWFQNRRMKWKK (Penetratin) (SEQ ID NO: 21); GRKKRRQRRRPPQC (Tat fragments 48-60) (SEQ ID NO: 22); GALFLGWLGAAGSTMGAWSQPKKKRKV (Signal sequence-based peptide) (SEQ ID NO: 23); LLIILRRRIRKQAHAHSK (PVEC) (SEQ ID NO: 24); GWTLNSAGYLLKINLKALAALAKKIL (Transportan) (SEQ ID NO: 25); KLALKLALKALKAALKLA (Amphiphilic model peptide) (SEQ ID NO: 26); RRRRRRRRR (Arg9) (SEQ ID NO: 27); KFFKFFKFFK (Bacterial cell wall-permeable peptide) (SEQ ID NO: 28); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37) (SEQ ID NO: 2) 9); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropine P1) (SEQ ID NO: 30); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO: 31); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO: 32); RRRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID NO: 33); ILPWKWPWWPWRR-NH2 (indollicidine) (SEQ ID NO: 34); AAVALLPAVLLALLAP (RFGF) (SEQ ID NO: 35); AALLPVLLAAP (RFGF analog) (SEQ ID NO: 36); and RKCRIVVIRVCR (bactenesin) (SEQ ID NO: 37).

[0419] Exemplary cationic groups include protonated amino groups derived from, for example, O-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxys, e.g., O(CH2) nAMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH (CH2CH2NH) n This includes, but is not limited to, CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0420] As used herein, the term “targeting ligand” refers to any molecule that results in enhanced affinity to a selective target, such as a cell, cell type, tissue, organ, region of the body, or compartment, such as a cellular compartment, tissue compartment, or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0421] 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 as GalNAc conjugates herein); D-mannose, polyvalent mannose, polyvalent lactose, N-acetylglucosamine, glucose, polyvalent glucose, polyvalent fucose, glycosylated polyamino acids, and lectins. The term polyvalent indicates the presence of more than one monosaccharide unit. Such monosaccharide subunits may be linked to one another via glycosidic linkages or to scaffold molecules.

[0422] Numerous folic acid and folic acid analogs suitable as ligands for the present invention are described in U.S. Patents 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the contents of which are incorporated herein by reference in their entirety.

[0423] As used herein, the terms “PK-modulating ligand” and “PK modulator” refer to molecules capable of modulating the pharmacokinetics of the compositions of the present invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and transthyretia-binding ligands (e.g., tetraiidothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligomer compounds containing numerous phosphorothioate linkages are also known to bind to serum proteins, and therefore, short oligomer compounds, such as oligonucleotides containing 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 those containing multiple phosphorothioate linkages in their backbone are also suitable for the present invention as ligands (e.g., as PK-modulating ligands). PK-modulating oligonucleotides may contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate linkages and / or phosphorodithioate linkages. In some embodiments, all internucleotide linkages within the PK-modulating oligonucleotide are phosphorothioate linkages and / or phosphorodithioate linkages. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable as PK-modulating ligands for the present invention. Binding to serum components (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.

[0424] When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties while others have different properties. For example, a ligand may have targeting properties, endosome lysis activity, or PK modulating properties. In a preferred embodiment, all ligands have different properties.

[0425] Ligands or tether ligands may be present on the monomer when the monomer is incorporated into the components of a single-stranded oligonucleotide. In some embodiments, the ligand may be incorporated after the "precursor" monomer has been incorporated into the components of the single-stranded oligonucleotide, via coupling to the "precursor" monomer. For example, a monomer having an amino-terminal tether (i.e., without an associated ligand), such as a monomer-linker-NH2 monomer, may be incorporated into the components of a single-stranded oligonucleotide. In a subsequent operation, i.e., after the precursor monomer has been incorporated into the components of the single-stranded oligonucleotide, a ligand having an electrophile, such as a pentafluorophenyl ester or aldehyde group, may be subsequently joined to the precursor monomer by coupling the electrophile of the ligand with the terminal nucleophile of the tether of the precursor monomer.

[0426] In another example, monomers having chemical groups suitable for participating in click chemistry reactions, such as azide or alkyne-terminated tethers / linkers, can be incorporated. In subsequent operations, i.e., after the precursor monomer has been incorporated into the chain, ligands having complementary chemical groups, such as alkynes or azides, can be joined to the precursor monomer by coupling the alkyne and azide together.

[0427] In some embodiments, ligands can be conjugated to a nucleobase, sugar moiety, or nucleoside linkage of a single-stranded oligonucleotide. Conjugation to purine nucleobases or their derivatives can occur at any position, including intra-ring and extra-ring atoms. In some embodiments, positions 2, 6, 7, or 8 of the purine nucleobase are joined to the conjugate moiety. Conjugation to pyrimidine nucleobases or their derivatives can also occur at any position. In some embodiments, positions 2, 5, and 6 of the pyrimidine nucleobase can be substituted at the conjugate moiety. When ligands conjugate to a nucleobase, preferred positions are those that do not interfere with hybridization, i.e., do not interfere with the hydrogen bonding interactions necessary for base pairing.

[0428] Conjugate to the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms in the sugar moiety that can conjugate to the conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also conjugate to the conjugate moiety, such as within a debasic residue. Nucleoside linkages can also possess a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiesters, phosphorothioates, phosphorodithioates, phosphoramidates, etc.), the conjugate moiety can be directly conjugated to the phosphorus atom, or to an O, N, or S atom bonded to the phosphorus atom. In the case of amine or amide-containing nucleoside linkages (e.g., PNA), the conjugate moiety can be conjugated to the nitrogen atom of the amine or amide, or to an adjacent carbon atom.

[0429] There are many methods for preparing oligonucleotide conjugates. Generally, oligonucleotides are joined to a conjugate site by contacting a reactive group on the oligonucleotide (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) with a reactive group on the conjugate site. In some embodiments, one reactive group is electrophilic and the other is nucleophilic.

[0430] For example, the electrophile may be a carbonyl-containing functional, and the nucleophile may be an amine or thiol. Methods for conjugating nucleic acids and related oligomeric compounds with or without linking groups are well described in 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.

[0431] Representative U.S. patents teaching the preparation of nucleic acid conjugates are incorporated herein by reference in their entirety: U.S. Patents 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; and 5,138,04 No. 5; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608 ,046;No.4,587,044;No.4,605,735;No.4,667,025;No.4,762,779;No.4, 789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830 No. 5,112,963; No. 5,149,782; No. 5,214,136; No. 5,245,022; No. 5,254, No. 469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,3 No. 17,098; No. 5,371,241; No. 5,391,723; No. 5,416,203; No. 5,451,463; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810 ; Same No. 5,574,142; Same No. 5,585,481; Same No. 5,587,371; Same No. 5,595,726; Same No. 5,597,6 No. 96; No. 5,599,923; No. 5,599,928; No. 5,672,662; No. 5,688,941; No. 5,71 No. 4,166; No. 6,153,737; No. 6,172,208; No. 6,300,319; No. 6,335,434; No. 6 , 335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031;This includes, but is not limited to, Articles No. 6,528,631 and No. 6,559,279.

[0432] In some embodiments, the single-stranded oligonucleotide further comprises one or more targeting ligands that target liver tissue. In some embodiments, at least one targeting ligand is a carbohydrate-based ligand. In some embodiments, the carbohydrate-based ligand is an ASGPR ligand. In one embodiment, at least one targeting ligand is a GalNAc-based conjugate.

[0433] In some embodiments, the carbohydrate-based ligand is one of the ligands listed in Table 2, Table 2A, Table 3, Table 3A, Table 4, or Table 4A of WO2015 / 006740, which are incorporated herein by reference in their entirety.

[0434] In some embodiments, linkers including branched linkers such as divalent or trivalent branched linkers that join these carbohydrate-based ligands include linkers(s) listed in Table 1 or Table 1A of WO2015 / 006740 and spacers(s) listed in Table 5, which are incorporated herein by reference in their entirety.

[0435] In some embodiments, the GalNAc-based conjugate is a GalNAc analog containing an S or N atom or a CH2 group in the glycosidic linkage, which converts a metabolically unstable glycosidic linkage into a metabolically stable glycosidic linkage, for example, in which the "O" in the glycosidic linkage is substituted with an S or N atom or a -CH2- group, as shown by the following scheme. [ka] The entire procedure is incorporated by reference, referring to the synthesis steps for these GalNAc analogs in Kandasamy et al., “Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathywas,” J. Med. Chem. 66:2506-23 (2023).

[0436] In some embodiments, the GalNAc-based conjugate is a GalNAc analog having one of the following structures: [Table 2-1] [Table 2-2]

[0437] The GalNAc analogs listed in the table above may be prepared using the method described in WO2015 / 006740, which is incorporated herein by reference in whole.

[0438] In some embodiments, the GalNAc-based conjugate has the following structure: [ka] [wherein n = 0 to 10 (e.g., 1 or 4). See Figures 4A and 4B of US2021 / 0123048A1, which are incorporated herein by reference in their entirety.] [ka] It is a GalNAc analog that possesses one of the following properties.

[0439] In a particular embodiment, the single-stranded oligonucleotide has the following structure: [ka] [In formula: L G Independently, for each occurrence, the ligand is a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. Z', Z'', Z''', and Z'''' are each independently O or S for each occurrence. Further comprises ligands having the following properties.

[0440] In a particular embodiment, the single-stranded oligonucleotide is of formula (II), (III), (IV), or (V): [ka] [In the formula, q 2A , q 2B , q 3A , q 3B q4 A , q 4B , q 5A , q 5B and q 5C Each occurrence independently represents a number between 0 and 20, in which case the repeating unit may be the same or different; Q and Q' are, independently, nonexistent or -(P) for each occurrence. 7 -Q 7 -R 7 ) p -T 7 -or -T 7 -Q 7 -T 7’ -BT 8’ -Q 8 -T 8 and; 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 , T4B , T 4A , T 5B , T 5C , T 7 , T 7’ , T 8 and T 8’ Each of these is, independently, for each occurrence, either absent, or 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 and; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Q 7 Q 8 and Q B Independently, for each occurrence, is either absent or an alkylene, a substituted alkylene, in which case one or more methylenes are O, S, S(O), SO2, N(R) N It may be interrupted or terminated by one or more of the following: C(R')=C(R'), C≡C, or C(O); T B and T B’ Each of these, independently, for each occurrence, is either absent or CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH2, CH2NH, or CH2O; R xThese are lipophilic [e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrene butyric 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 phenoxa These include gin, vitamins (e.g., folic acid, vitamin A, vitamin E, biotin, pyridoxal), peptides, carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides), endosomal lysants, steroids (e.g., ubaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-derived lithocholic acid), or cationic lipids; R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 Each of these, independently, for each occurrence, is either absent or consists of NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO [ka] Or it is a heterocycline; L 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5CEach of these is, independently, a carbohydrate (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides) for each occurrence; R' and R'' are, independently, H, C1-C6 alkyl, OH, SH, or N(R) N )2; R N Independently, for each occurrence, these are H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; R a is H or an amino acid side chain; Z', Z'', Z''', and Z'''' are each independently O or S for each occurrence; [p independently represents 0 to 20 for each occurrence.] It contains ligands.

[0441] As discussed above, since ligands can be conjugated to single-stranded oligonucleotides via linkers or carriers, and since linkers or carriers can contain branched linkers, single-stranded oligonucleotides can contain multiple ligands via the same or different skeletal junctions to the carrier, or via branched linkers. For example, the branching point of a branched linker can be a divalent, trivalent, tetravalent, pentavalent, or hexavalent atom, or a group exhibiting multiple such valencies. In certain embodiments, the branching point is -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC, [wherein Q is independently, for each occurrence, H or optionally substituted alkyl]. In other embodiments, the branching point is glycerol or a glycerol derivative.

[0442] In a particular embodiment, the ASGPR ligand conjugated to a single-strand oligonucleotide is one or more GalNAc derivatives linked by a divalent or trivalent branched linker.

[0443] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0444] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0445] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0446] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0447] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0448] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0449] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0450] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0451] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0452] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0453] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0454] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0455] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands. Exemplary ligand monomers

[0456] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0457] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0458] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0459] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0460] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0461] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0462] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0463] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0464] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0465] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0466] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0467] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0468] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0469] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0470] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0471] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0472] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0473] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0474] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0475] In a particular embodiment, the single-stranded oligonucleotide of the present invention is [ka] It contains monomers.

[0476] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0477] In some embodiments, L 2A and L 2B Both are the same. In some embodiments, L 2A and L2B The two are different.

[0478] In some embodiments, L 3A and L 3B Both are the same. In some embodiments, L 3A and L 3B The two are different.

[0479] In some embodiments, L 4A and L 4B Both are the same. In some embodiments, L 4A and L 4B The two are different.

[0480] In some embodiments, L 5A , L 5B and L 5C All of them are the same. In some embodiments, L 5A , L 5B and L 5C The two are the same. In some embodiments, L 5A and L 5B They are the same. In some embodiments, L 5A and L 5C They are the same. In some embodiments, L 5B and L 5C They are the same.

[0481] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0482] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0483] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0484] In a particular embodiment, a single-stranded oligonucleotide is [ka] [In the formula, Y is either O or S, and n is between 1 and 6.] It contains monomers.

[0485] In a particular embodiment, a single-stranded oligonucleotide is [ka] [In the formula, Y is O or S, n is 1 to 6, R is hydrogen or nucleic acid, and R' is nucleic acid] It contains monomers.

[0486] In a particular embodiment, a single-stranded oligonucleotide is [ka] [In the formula, Y is either O or S, and n is between 1 and 6.] It contains monomers.

[0487] In a particular embodiment, the single-stranded oligonucleotide has the following structure: [ka] [In the formula, Y is O or S, n is 2 to 6, x is 1 to 6, and A is H or a phosphate linkage] It contains monomers.

[0488] In some embodiments, single-stranded oligonucleotides are [ka] It contains at least one, two, three, or four monomers.

[0489] In some embodiments, single-stranded oligonucleotides are [ka] [In the formula, X is either O or S] It contains monomers.

[0490] In some embodiments, single-stranded oligonucleotides are [ka] [In the formula, x is between 1 and 12] It contains monomers.

[0491] In some embodiments, single-stranded oligonucleotides are [ka] [In the formula, R is either OH or NHCOCH3] It contains monomers.

[0492] In a particular embodiment, a single-stranded oligonucleotide is [ka] [In the formula, R is either OH or NHCOCH3] It contains monomers.

[0493] In a particular embodiment, a single-stranded oligonucleotide is [ka] [In the formula, R is either O or S] It contains monomers.

[0494] In a particular embodiment, a single-stranded oligonucleotide is [ka] [In the formula, R is either OH or NHCOCH3] It contains monomers.

[0495] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0496] In some embodiments, single-stranded oligonucleotides are [ka] [In the formula, R is either OH or NHCOCH3] It contains monomers.

[0497] In some embodiments, single-stranded oligonucleotides are [ka] [In the formula, R is either OH or NHCOCH3] It contains monomers.

[0498] In some embodiments, single-stranded oligonucleotides are [ka] [In the formula, R is either OH or NHCOCH3] It contains monomers.

[0499] In a particular embodiment, a single-stranded oligonucleotide is [ka] [In the formula, R is either OH or NHCOCH3] It contains monomers.

[0500] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains monomers.

[0501] In the monomers described above, X and Y are, independently for each occurrence, H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, a -P(Z')(Z'')O-nucleoside, a -P(Z')(Z'')O-oligonucleotide, a lipid, PEG, a steroid, a polymer, a nucleotide, a nucleoside, or an oligonucleotide; and Z' and Z'' are, independently for each occurrence, O or S.

[0502] In some embodiments, single-stranded oligonucleotides are [ka] It is conjugated with its ligand.

[0503] In a particular embodiment, a single-stranded oligonucleotide is [ka] It contains ligands.

[0504] In a particular embodiment, a single-stranded oligonucleotide is [ka] This includes monomers. The synthesis of the above ligands and monomers is described, for example, in U.S. Patent No. 8,106,022, which is incorporated herein by reference in its entirety.

[0505] In a particular embodiment, at least one of the ligands conjugated to the single-stranded oligonucleotide is a lipophilic moiety.

[0506] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by its octanol-water partition coefficient, logKow Therefore, K ow The logK of a two-phase system at equilibrium is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of the chemical, calculated using first-principles or experimental methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the full contents of which are incorporated herein by reference). It provides a thermodynamic measurement of a substance's tendency to prefer non-aqueous or oily environments to water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical substance has a logK of 100. ow If logK is greater than 0, the property is lipophilic. Typically, the lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow It has. For example, the logK of 6-aminohexanol ow For example, it is predicted to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol) carbamate ow It is predicted to be 10.7.

[0507] The lipophilicity of a molecule can be altered with respect to its functional groups. For example, the addition of a hydroxyl group or amine group to the terminal of a lipophilic moiety can change the partition coefficient (e.g., logK) of the lipophilic moiety. ow The value can be increased or decreased.

[0508] Alternatively, the hydrophobicity of a single-stranded oligonucleotide conjugated to one or more lipophilic moieties can be measured by its protein-binding properties. For example, the unbound fraction in a plasma protein-binding assay of a single-stranded oligonucleotide can be determined to positively correlate with the relative hydrophobicity of the single-stranded oligonucleotide, which may positively correlate with the silencing activity of the single-stranded oligonucleotide.

[0509] In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the single-stranded oligonucleotide, as measured by the fraction of unbound single-stranded oligonucleotides in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5, for enhanced in vivo delivery of the single-stranded oligonucleotide.

[0510] Therefore, conjugation of a lipophilic moiety to one or more internal positions of a single-stranded oligonucleotide provides optimal hydrophobicity to enhance the in vivo delivery of the single-stranded oligonucleotide.

[0511] In certain embodiments, the lipophilic moiety is an aliphatic, cyclic, or polycyclic compound such as aliphatic, alicyclic, or polycyclic alicyclic, e.g., a steroid (e.g., a sterol) or a linear or branched aliphatic hydrocarbon. The lipophilic moiety generally comprises a hydrocarbon chain that can be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, e.g., an oxygen atom or a nitrogen atom. Such a lipophilic aliphatic moiety may be saturated or unsaturated C4-C 30 Hydrocarbon chains (for example, C6~C 18 Hydrocarbons or C 14 ~C 24 Hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters and fatty diamides of fatty acids), terpenes (e.g., C 10 Terpenes, C 15 Sesquiterpenes, C 20 Diterpenes, C 30 Triterpenes, and C 40 This includes, but is not limited to, tetraterpenes and other polycyclic alicyclic hydrocarbons. For example, the lipophilic portion is C4-C 30 Hydrocarbon chains (for example, C4~C 30 It may contain alkyl or alkenyl compounds. In some embodiments, the lipophilic portion is saturated or unsaturated C6-C6. 18Hydrocarbon chains (e.g., linear C6~C) 18 (Alkyl or alkenyl) or saturated or unsaturated C 14 ~C 24 Hydrocarbon chains (e.g., linear C) 14 ~C 24 It contains alkyl or alkenyl compounds. In one embodiment, the lipophilic portion is saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C) 16 (Alkyl or alkenyl) or saturated or unsaturated C 22 Hydrocarbon chains (e.g., linear C) 22 Contains alkyl or alkenyl compounds.

[0512] The lipophilic moiety may be joined to the single-stranded oligonucleotide by any method known in the art via a functional group already present in the lipophilic moiety or introduced into the single-stranded oligonucleotide, such as a hydroxyl group (e.g., -CO-CH2-OH). Functional groups already present in the lipophilic moiety or introduced into the single-stranded oligonucleotide include, but are not limited to, hydroxyls, amines, carboxylic acids, sulfonic acids, phosphoric acids, thiols, azides, and alkynes.

[0513] Conjugation between a single-chain oligonucleotide and a lipophilic moiety can occur, for example, through the formation of an ether or carboxylic acid or carbamoyl ester linkage between a hydroxyl group and an alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., linear or branched; and saturated or unsaturated). The alkyl group R can be butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl groups, etc.

[0514] In some embodiments, the lipophilic portion is conjugated to a single-chain oligonucleotide via a linker, the linker containing an ether, thioether, urea, carboxylic acid, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, click reaction product (e.g., triazole from azido-alkyne cycloaddition), or carbamate.

[0515] In another embodiment, the lipophilic portion is a steroid such as a sterol. The steroid is a polycyclic compound containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. "Cholesterol derivative" refers to a cholesterol-derived compound, for example, by substitution, addition, or removal of substituents.

[0516] In another embodiment, the lipophilic portion is the aromatic portion. In this context, the term "aromatic" broadly refers to monocyclic and polycyclic aromatic hydrocarbons. The aromatic group is a C6-C6 molecule containing 1-3 aromatic rings, which may be substituted as appropriate. 14 The term "heteroaryl" includes, but is not limited to, aryl groups; and "aralkyl" or "arylalkyl" groups, which include an aryl group covalently bonded to an alkyl group, either of which may independently be substituted or unsubstituted as appropriate; and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14π electrons shared in a cyclic arrangement; and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0517] The “substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic groups used herein have non-hydrogen substitutions between 1 and about 4, preferably between 1 and about 3, more preferably 1 or 2. Preferred substitutions include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkylaryl (alkaryl), aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0518] In some embodiments, the lipophilic moiety is an aralkyl group, for example, a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety binds to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, blood vessels, or cellular proteins. In certain embodiments, the structural features of the aralkyl group facilitate binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulins, or α-1-glycoproteins.

[0519] In certain embodiments, the ligand is naproxene or a structural derivative of naproxene. The synthesis procedure for naproxene can be found in U.S. Patents 3,904,682 and 4,009,197, which are incorporated herein by reference in their entirety. Naproxene has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, and its structure is [ka] That is the case.

[0520] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. A procedure for the synthesis of ibuprofen can be found in U.S. Patent No. 3,228,831, which is incorporated herein by reference in its entirety. The structure of ibuprofen is: [ka] That is the case.

[0521] Further exemplary aralkyl groups are exemplified in U.S. Patent No. 7,626,014, which is incorporated herein by reference in whole.

[0522] In another embodiment, a preferred lipophilic moiety includes lipids, cholesterol, retinoic acid, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0523] In some embodiments, the lipophilic portion is C6~C 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexanoenic acid, vitamin A, vitamin E, cholesterol, etc.) or C6~C 30These are alcohols (for example, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.). In one example, the lipophilic portion is docosahexaenoic acid.

[0524] In certain embodiments, more than one lipophilic moiety may be incorporated into a single-stranded oligonucleotide, particularly if the lipophilic moiety has low lipophilicity or hydrophobicity. In one embodiment, two or more lipophilic moieties are incorporated into the same chain of a single-stranded oligonucleotide. In one embodiment, each chain of a single-stranded oligonucleotide has one or more incorporated lipophilic moieties. In one embodiment, two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage) of the single-stranded oligonucleotide. This can be achieved, for example, by conjugating two or more lipophilic moieties via a carrier, and / or by conjugating two or more lipophilic moieties via a branched linker, and / or by conjugating two or more lipophilic moieties via one or more linkers by one or more linkers that sequentially link lipophilic moieties.

[0525] The lipophilic moiety can be conjugated to a single-stranded oligonucleotide via direct linking to the ribosaccharide of the single-stranded oligonucleotide. Alternatively, the lipophilic moiety can be conjugated to a single-stranded oligonucleotide via a linker or carrier.

[0526] In a particular embodiment, the lipophilic moiety may be conjugated to a single-strand oligonucleotide via one or more linkers (tethers).

[0527] In one embodiment, the lipophilic moiety is conjugated to a single-strand oligonucleotide via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, click reaction product (e.g., triazole from azido-alkyne cyclic addition), or carbamate.

[0528] definition Unless otherwise noted, the nomenclature, procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, 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. Certain such techniques and procedures are incorporated herein in their entirety by reference for all purposes, for example, in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington DC, 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A Laboratory Manual,” 2 nd See Edition, Cold Spring Harbor Laboratory Press, 1989. Where permitted, all patents, applications, published applications and other publications and other data referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0529] Unless otherwise specified, the following terms have the following meanings.

[0530] As used herein, the term “target nucleic acid” refers to any nucleic acid molecule whose expression or activity can be modulated 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), as well as cDNA and miRNA derived from such RNA. For example, a target nucleic acid may be a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a particular disorder or disease condition. In some embodiments, a target nucleic acid may be a nucleic acid molecule derived from an infectious pathogen.

[0531] As used herein, the term “iRNA” refers to factors that mediate targeted cleavage of RNA transcripts. These factors associate with a cytoplasmic multiprotein complex known as the RNAi-inducible silencing complex (RISC). Factors effective in inducing RNA interference are also referred herein to as siRNA, RNAi agents, or iRNA agents. These terms may be used interchangeably herein. As used herein, the term iRNA includes microRNA and premicroRNA. Furthermore, as used herein, “compound” or “compounds” of the present invention also refers to iRNA agents and may be used interchangeably with iRNA agents.

[0532] The iRNA agent must contain a region with sufficient homology to the target gene, be of sufficient length with respect to nucleotides, and the iRNA agent or a fragment thereof must be capable of mediating the downregulation of the target gene. (For ease of explanation, the terms nucleotide or ribonucleotide may be used herein in reference to one or more monomeric subunits of the iRNA agent. It should be understood herein that the use of the terms “ribonucleotide” or “nucleotide” may also refer to a modified nucleotide or substitute substitution region at one or more positions in the case of modified RNA or nucleotide substitutes.) Therefore, the iRNA agent is or contains a region that is at least partially, and in some embodiments, fully complementary to the target RNA. While complete complementarity between the iRNA agent and the target is not required, there must be sufficient correspondence to enable the iRNA agent or its cleavage products 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. While complete complementarity is often desired, particularly in the antisense strand, some embodiments may include one or more mismatches, such as 6, 5, 4, 3, 2, or fewer, especially in the antisense strand (with respect to the target RNA). The sense strand only needs to be sufficiently complementary to the antisense strand in order to maintain the overall double-stranded nature of the molecule.

[0533] iRNA agents include molecules long enough to induce an interferon response [which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)], and molecules short enough not to induce an interferon response (which can also be cleaved by Dicer and / or enter RISC), e.g., molecules small enough to enter RISC, e.g., molecules similar to the cleavage products by Dicer. Molecules short enough not to induce an interferon response are referred to herein as siRNA agents or shorter iRNA agents. As used herein, “siRNA agents or shorter iRNA agents” refers to iRNA agents short enough not to induce a harmful interferon response in human cells, e.g., double-stranded RNA agents or single-stranded RNA agents having a double-stranded region of less than 60, 50, 40, or 30 nucleotide pairs. siRNA agents, or their cleavage products, can downregulate target genes, for example, by inducing RNAi with respect to target RNA, and the target may include endogenous or pathogenic target RNA.

[0534] As used herein, “single-stranded oligonucleotide” or “single-stranded iRNA agent” is an oligonucleotide or iRNA agent consisting of one molecule. A single-stranded oligonucleotide may contain a double-stranded region formed by intra-chain pairing, which may be, for example, a hairpin structure, a dumbbell structure, or a panhandle structure, or may contain such a structure. A single-stranded oligonucleotide or iRNA agent may be antisense against a target molecule. A single-stranded oligonucleotide or iRNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of target mRNA. A single-stranded oligonucleotide or iRNA agent is at least 14 nucleotides long, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides long. In certain embodiments, the length is less than 200, 100, or 60 nucleotides. In certain embodiments, a single-stranded oligonucleotide contains two oligonucleotides linked by a linking group.

[0535] A loop refers to a region in a double helix that is not paired with the opposing nucleotide when an oligonucleotide or iRNA strand forms a base pair with another strand or another part of the same strand.

[0536] A hairpin oligonucleotide or iRNA agent may have a double-stranded region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The double-stranded region may be 200, 100, or 50 or less in length. In certain embodiments, the double-stranded region ranges in length from 15–30, 17–23, 19–23, and 19–21 nucleotide pairs. The hairpin may have a single-stranded overhang or terminal unpaired region at its 3' end in some embodiments, or at the antisense side of the hairpin in certain embodiments. In some embodiments, the overhang is 2–3 nucleotides long.

[0537] As used herein, "double-stranded oligonucleotide," "double-stranded nucleic acid agent," or "double-stranded (ds) iRNA agent" refers to an oligonucleotide, nucleic acid agent, or iRNA agent containing one or more, possibly two, strands, in which interstrand hybridization can form a region of a double-stranded structure.

[0538] As used herein, the terms “activity,” “siRNA activity,” or “RNAi activity” refer to gene silencing by oligonucleotides, nucleic acid agents, or iRNA agents.

[0539] As used herein, “gene silencing” by oligonucleotides, nucleic acid agents, or iRNA agents means that the mRNA level in a cell containing the target gene is reduced to at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, and any integer between 5% and 100%. In a preferred embodiment, the mRNA level is reduced to at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, and any integer between 5% and 100%.

[0540] As used herein, the term “modulate gene expression” means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated to be greater or less than the expression, level, or activity observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the term “modulate” is not limited to this definition.

[0541] Gene expression modulation as used herein occurs when the level of gene expression, or RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, differs from that observed in the absence of siRNA by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2x, 3x, 4x, or 5x. The percentage and / or multiplier of this difference is, for example,

number

number

[0542] As used herein, the terms “inhibit,” “downregulate,” or “reduce” in relation to gene expression mean that the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced to what is observed in the absence of the modulator. Downregulation of gene expression occurs when the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably 100% (i.e., no gene expression) compared to the corresponding unmodulated control.

[0543] As used herein, the terms “increased” or “upregulated” in relation to gene expression mean that the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased compared to what is observed in the absence of the modulator. Upregulated gene expression occurs when the level of gene expression, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased by at least 10% compared to the corresponding unmodulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1x, 1.25x, 1.5x, 1.75x, 2x, 3x, 4x, 5x, 10x, 50x, 100x or more.

[0544] As used herein, the terms “increased” or “increasing” generally mean an increase of a statistically significant amount. To avoid any doubt, “increased” means an increase of at least 10% compared to a baseline level, for example, an increase of 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%, or an increase of at least about 90%, or up to 100%, or any increase between 10% and 100% compared to a baseline level, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a baseline level, or any increase between 2 times and 10 times or more compared to a baseline level.

[0545] As used herein, the terms “reduced” or “decrease” generally mean a statistically significant decrease. However, to avoid any doubt, “reduced” means a decrease of at least 10% compared to a reference level, e.g., 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% (i.e., a level that does not exist compared to a reference sample), or any decrease between 10% and 100% compared to a reference level.

[0546] Double-stranded nucleic acid agents comprise two oligonucleotide chains that are sufficiently complementary to hybridize to form a double-stranded structure. Generally, the double-stranded structure has a base pair length of between 8 and 30, between 15 and 30, between 18 and 25, between 19 and 24, or between 19 and 21. In some embodiments, long double-stranded nucleic acid agents with a base pair length between 25 and 30 are preferred. In some embodiments, short double-stranded nucleic acid agents with a base pair length between 10 and 15 are preferred. In another embodiment, the double-stranded nucleic acid agent is at least 21 nucleotides long.

[0547] As used herein, the terms “antisense chain” or “antisense oligonucleotide” refer to an oligomeric compound that is substantially or 100% complementary to the target sequence of interest. The term “antisense chain” includes the antisense region of both an oligomeric compound formed from two separate chains and a monomolecular oligomeric compound that can form a hairpin or dumbbell-shaped structure. The terms “antisense chain” and “guide chain” are used interchangeably herein.

[0548] The term "sense strand" refers to an oligomeric compound having a nucleoside sequence that is all or part identical to a target sequence, such as a messenger RNA or DNA sequence. The terms "sense strand" and "passenger strand" are used interchangeably herein.

[0549] "Specifically hybridizable" and "complementary" mean that a nucleic acid can form hydrogen bonds (or more) with another nucleic acid sequence in either a conventional Watson-Crick configuration or a non-conventional configuration. With respect to the nucleic acid molecules of the present invention, the binding free energy between the nucleic acid molecule and its complementary sequence is sufficient to enable the relevant function of the nucleic acid, 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). Complementarity percentage refers to the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Full complementarity" or 100% complementarity means that all consecutive residues in a nucleic acid sequence can form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. Less than full complementarity refers to a situation where some, but not all, nucleoside units in the two strands can form hydrogen bonds with each other. "Substantial complementarity" refers to polynucleotide chains that exhibit 90% or more complementarity, excluding regions of the polynucleotide chain that are selected to be non-complementary, such as overhangs. Specific binding requires a degree of complementarity sufficient to avoid nonspecific binding of the oligomeric compound to the non-target sequence under the conditions under which specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic procedures, or under the conditions under which the assay is performed in the case of in vitro assays. The non-target sequence is typically at least 5 nucleotides different.

[0550] In some embodiments, the double-stranded region of the double-stranded nucleic acid agent is equal to the length of 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, or at least the length of 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.

[0551] In some embodiments, the first oligonucleotide of the double-stranded nucleic acid agent is equal in length to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0552] In some embodiments, the second oligonucleotide of the double-stranded nucleic acid agent is equal to 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.

[0553] In one embodiment, the first and second oligonucleotides of the double-stranded nucleic acid agent are each 15 to 30 nucleotides in length.

[0554] In one embodiment, the first and second oligonucleotides of the double-stranded nucleic acid agent are each 19 to 25 nucleotides in length.

[0555] In one embodiment, the first and second oligonucleotides of the double-stranded nucleic acid agent are each 21 to 23 nucleotides in length.

[0556] In some embodiments, one oligonucleotide has at least one stretch of 1 to 5 single-stranded nucleotides in its double-stranded region. "Stretch of single-stranded nucleotides in its double-stranded region" means that there is at least one nucleotide base pair at both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in its double-stranded region. If both strands have stretches of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in their double-stranded regions, such single-stranded nucleotides may be arranged so that they do not have single-stranded nucleotides opposite each other (e.g., mismatched stretches), or so that the second oligonucleotide does not have single-stranded nucleotides opposite to the single-stranded nucleotides of the first oligonucleotide, and vice versa (e.g., single-stranded loops). In some embodiments, the single-stranded nucleotides are located within 8 nucleotides from either end, for example, within 8 nucleotides from either the 5' or 3' end of the complementary region between two oligonucleotides, for example, within 8, 7, 6, 5, 4, 3, or 2 nucleotides.

[0557] In one embodiment, the double-stranded nucleic acid agent includes a single-stranded projection at at least one of its ends. In one embodiment, the single-stranded projection is 1, 2, or 3 nucleotides long.

[0558] In one embodiment, the second oligonucleotide of the double-stranded nucleic acid agent is 21 nucleotides long, and the first oligonucleotide is 23 nucleotides long, where the first and second oligonucleotides form a double-stranded region of 21 consecutive base pairs, each having a single-stranded projection 2 nucleotides longer at its 3' end.

[0559] In some embodiments, each oligonucleotide of the double-stranded nucleic acid agent has a ZXY structure as described in PCT Publication No. 2004080406, which is incorporated herein by reference in its entirety.

[0560] In a particular embodiment, two nucleotide sequences can be linked together to form a long chain. The two nucleotide sequences are (N) n The oligonucleotides may be linked together by an oligonucleotide linker, which includes, but is not limited to, a sequence of oligonucleotides (wherein N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3 to 10, for example, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4 (wherein N is a modified or unmodified nucleotide, and R is a modified or unmodified purine nucleotide). Some of the nucleotides in the linker may be involved in base-pair interactions with other nucleotides in the linker. The two nucleotide sequences may be linked together by a non-nucleotide-based linker, for example, a linker described herein. It will be understood by those skilled in the art that any oligonucleotide chemical modification or variation described herein may be used for the oligonucleotide linker.

[0561] In certain embodiments, the two strands specifically hybridize if there is sufficient complementarity to avoid nonspecific binding of the antisense compound to a non-target nucleic acid sequence under conditions where specific binding is desired, i.e., under physiological conditions in the case of an in vivo assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay.

[0562] As used herein, “strict hybridization conditions” or “strict conditions” refer to conditions under which an antisense compound hybridizes to its target sequence but to a minimum number of other sequences. Strict conditions are sequence-dependent and vary in different situations, and the “strict conditions” under which an antisense compound hybridizes to its target sequence are determined by the properties and composition of the antisense compound, as well as the assay in which they are studied.

[0563] It is understood in the art that the incorporation of nucleotide affinity modifications can tolerate more mismatches compared to unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences. Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides, or between oligonucleotides and target nucleic acids, by determining the melting temperature (Tm), for example. Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, using the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443), those skilled in the art can evaluate the ability of nucleotide modifications to increase the melting temperature of RNA:DNA double helix.

[0564] Single-stranded oligonucleotides may contain a phosphorus-containing group at the 5' end of the nucleotide sequence. The phosphorus-containing group at the 5' end may be a 5'-terminal phosphate (5'-P), a 5'-terminal phosphorothioate (5'-PS), a 5'-terminal phosphorodithioate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl( [ka] ) may be. If the phosphorus-containing group at the 5' terminus is a vinyl phosphonate (5'-VP), then 5'-VP is a 5'-E-VP isomer (i.e., trans-vinyl phosphate, [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ), or a mixture thereof.

[0565] In one embodiment, the single-stranded oligonucleotide is a nucleotide sequence (e.g., Z 1 and / or Z 2 ) contains a phosphorus-containing group at its 5' end.

[0566] In one embodiment, the single-stranded oligonucleotide is a sequence of at least one nucleotide (e.g., Z 1 and / or Z 2 ) includes 5'-P.

[0567] In one embodiment, the single-stranded oligonucleotide is a sequence of at least one nucleotide (e.g., Z 1 and / or Z 2 ) contains 5'-PS.

[0568] In one embodiment, the single-stranded oligonucleotide is a sequence of at least one nucleotide (e.g., Z 1 and / or Z 2 ) contains 5'-VP. In one embodiment, the single-stranded oligonucleotide has at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ) contains 5'-E-VP. In one embodiment, the single-stranded oligonucleotide has at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ) includes 5'-Z-VP.

[0569] In one embodiment, the single-stranded oligonucleotide is a sequence of at least one nucleotide (e.g., Z 1 and / or Z 2 ) includes 5'-PS2.

[0570] In one embodiment, the single-stranded oligonucleotide is a sequence of at least one nucleotide (e.g., Z 1 and / or Z 2 ) contains 5'-deoxy-5'-C-malonyl.

[0571] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of a single-stranded oligonucleotide is modified. For example, if 50% of a single-stranded oligonucleotide is modified, all 50% of the nucleotides present in the single-stranded oligonucleotide contain the modifications described herein.

[0572] In one embodiment, the Z of a single-stranded oligonucleotide 1 and Z 2 Each nucleotide can be independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-alla-F.

[0573] In one embodiment, the nucleotide sequence of a single-stranded oligonucleotide (for example, Z 1 and / or Z 2 ) contains at least two different modifications.

[0574] In one embodiment, the single-stranded oligonucleotide does not contain 2'-F modification.

[0575] In one embodiment, a single-stranded oligonucleotide comprises one or more blocks of phosphorothioate or methylphosphonate nucleotide linkages. In one example, the single-stranded oligonucleotide comprises one block of two phosphorothioate or methylphosphonate nucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate nucleotide linkages are separated by 16 to 18 phosphate nucleotide linkages.

[0576] In one embodiment, a nucleotide sequence (for example, Z1 and / or Z 2 The nucleotide at position 1 of the 5' end of ) is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, the nucleotide sequence (e.g., Z 1 and / or Z 2 At least one of the first, second, and third base pairs from the 5' end of ) is an AU base pair.

[0577] In one embodiment, the single-stranded oligonucleotide is 100% complementary to the target RNA, hybridizes with it, and inhibits its expression by RNA interference. In another embodiment, the single-stranded oligonucleotide 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.

[0578] In some embodiments of this specification, single-stranded oligonucleotides capable of inhibiting the expression of a target gene are provided. The single-stranded oligonucleotide contains at least one thermally unstable nucleotide.

[0579] Thermally unstable nucleotides are, for example, nucleotide sequences with a length of 21 nucleotides (e.g., Z 1 and / or Z 2 This can occur between positions 14 and 17 of the 5' end. The nucleotide sequence may contain at least two modified nucleic acids smaller than the sterically required 2'-OMe modification. Preferably, the two modified nucleic acids smaller than the sterically required 2'-OMe are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end.

[0580] In some embodiments, the single-stranded oligonucleotide is given by formula (II): 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3' (II) [In formula: i and j are independently either 0 or 1; p and q are independently between 0 and 6; Each Na independently contains 0 to 25 modified nucleotides, and each sequence represents an oligonucleotide sequence containing at least two nucleotides with different modifications; Each Nb independently represents an oligonucleotide sequence containing 1, 2, 3, 4, 5, or 6 modified nucleotides. Each np and each nq independently represents an overhanging nucleotide; Here, Nb and Y do not have the same modifications; Here, XXX, YYY, and ZZZ each independently represent a single motif consisting of three identical modifications on three consecutive nucleotides. It contains sequences that can be represented by [this].

[0581] In some embodiments, the single-stranded oligonucleotide contains 2'-F modifications of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one example, the single-stranded oligonucleotide contains 2'-F modifications of 9 or 10.

[0582] A single-stranded oligonucleotide may further contain at least one phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage. Modification by phosphorothioate nucleotide linkage or methylphosphonate nucleotide linkage may occur at any nucleotide of the single-stranded oligonucleotide. For example, modification by nucleotide linkage may occur at any nucleotide on at least one nucleotide sequence; each modification by nucleotide linkage may occur in an alternating pattern on at least one nucleotide sequence.

[0583] In some embodiments, the compounds of the present invention disclosed herein are miRNA mimetic. In one design, the miRNA mimetic is a double-stranded molecule (e.g., having a double-stranded region between about 16 and about 31 nucleotides in length) and contains one or more sequences that are identical to the mature strand of a given miRNA. A double-stranded miRNA mimetic has a similar design to that described above for a double-stranded iRNA. In some embodiments, the miRNA mimetic comprises a double-stranded region of nucleotides between 16 and 31 and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counted from the 5' end of the sense strand) and all C and U; and the antisense strand modifications may include 2'F modifications of all C and U, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide ligatures associated with 2 nucleotide 3' overhangs.

[0584] In some embodiments, the compounds of the present invention disclosed herein are antimirs. In some embodiments, the compounds of the present invention comprise at least two antimirs covalently or noncovalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linker described herein. The terms “antimir,” “microRNA inhibitor,” or “miR inhibitor” are synonymous and refer to oligonucleotides or modified oligonucleotides that inhibit the activity of a particular miRNA. Inhibitors can employ a variety of configurations, including single-stranded, double-stranded (RNA / RNA or RNA / DNA double-stranded), and hairpin designs. Generally, a microRNA inhibitor comprises one or more sequences or a portion of sequences that are complementary or partially complementary to the mature strand (or strand) of the target miRNA. Furthermore, a miRNA inhibitor may also comprise an additional sequence located at 5' and 3' of the sequence that is the reverse complementary sequence of the mature miRNA. The additional sequence may be the reverse complementary sequence of a sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA originates, or the additional sequence may be any sequence (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences are arbitrary sequences that can form a hairpin. Thus, in some embodiments, the sequence that is the reverse complementary sequence of the miRNA is flanked by hairpin structures on the 5' and 3' ends. MicroRNA inhibitors, in the case of double-stranded microRNAs, can include mismatches between nucleotides on opposite strands. Furthermore, microRNA inhibitors can be ligated with a conjugate moiety to facilitate the uptake of the inhibitor into the cell.

[0585] MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13: 723-730 (2007), and WO2007 / 095387 and WO2008 / 036825, the entire contents of which are incorporated herein by reference. Those skilled in the art can select sequences from databases for desired miRNAs and design inhibitors useful for the methods disclosed herein.

[0586] In some embodiments, the compounds of the present invention disclosed herein are antagomirs. In some embodiments, the compounds of the present invention comprise at least two antagomirs covalently or noncovalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linker described herein. Antagomirs are RNA-like oligonucleotides and have various modifications for RNAse protection and pharmacological properties, such as enhanced uptake into tissues and cells. They differ from ordinary RNA, for example, by complete 2'-O-methylation of sugars, phosphorothioate intersaccharide links, and, for example, a cholesterol moiety at the 3' terminus. In preferred embodiments, an antagomir comprises 2'-O-methylation of all nucleotides, a cholesterol moiety at the 3' terminus, two phosphorothioate intersaccharide links at the first two positions of the 5' terminus, and four phosphorothioate links at the 3' terminus of the molecule. Antagomirs can be used to efficiently suppress endogenous miRNAs by forming a double helix containing the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which is expressly incorporated herein in its entirety by reference.

[0587] Recent studies have also found that dsRNAs can activate gene expression, a mechanism known as "small RNA-mediated gene activation" or RNAa (activated RNA). See, for example, Li, LC et al. Proc Natl Acad Sci US A. (2006), 103(46):17337-42, and Li LC (2008), "Small RNA-Mediated Gene Activation". RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press. ISBN 978-1-904455-25-7. dsRNAs targeting gene promoters have been shown to induce potent transcriptional activation of related genes. Endogenous miRNAs that cause RNAa have also been found in humans. Check E. Nature (2007). 448 (7156): 855-858.

[0588] Another surprising observation is the long-lasting nature of RNAa-mediated gene activation. Induction of gene expression has been observed to last for more than 10 days. The prolonged effect of RNAa may be due to epigenetic changes at the dsRNA target site. In some embodiments, RNA activators can increase gene expression. In some embodiments, increased gene expression inhibits viability, growth, development, and / or regeneration.

[0589] Accordingly, in some embodiments, the compounds of the present invention disclosed herein are activated RNAs. In some embodiments, the compounds of the present invention comprise at least two activated RNAs covalently or noncovalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linker described herein.

[0590] Accordingly, in some embodiments, the compounds of the present invention disclosed herein are triple-helix-forming oligonucleotides (TFOs). In some embodiments, the compounds of the present invention comprise at least two TFOs covalently or non-covalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linker described herein. Recent studies have shown that triple-helix-forming oligonucleotides can be designed to sequence-specifically recognize and bind to polypurine / polypyrimidine regions in double-stranded helix DNA. These recognition rules are outlined in Maher III, LJ, et al., Science (1989) vol. 245, pp 725-730; Moser, HE, et al., Science (1987) vol. 238, pp 645-630; Beal, PA, et al., Science (1992) vol. 251, pp 1360-1363; Conney, M., et al., Science (1988) vol. 241, pp 456-459 and Hogan, ME, et al., EP Publication 375408. Modifications of oligonucleotides, such as the introduction of intercalators and substitution of sugar-intersaccharide bonds, and optimization of binding conditions (pH and cation concentration) have helped overcome obstacles inherent to TFO activity, such as charge repulsion and instability, and it has recently been shown that synthetic oligonucleotides can be targeted to specific sequences (see Seidman and Glazer, J Clin Invest 2003;1 12:487-94 for a recent review). Generally, triple-chain-forming oligonucleotides correspond to sequences such as the following: Oligo3'-AGGT Double-stranded 5'-AGCT Double-stranded 3'-TCGA

[0591] However, A-AT and G-GC triplets have been shown to have the highest triple helix stability (Reither and Jeltsch, BMC Biochem, 2002, Septl2, Epub). The same authors have shown that TFOs designed according to the A-AT and G-GC rules do not form nonspecific triple helices, indicating that triple helix formation is indeed sequence-specific.

[0592] Therefore, a triple-helix-forming sequence can be devised for any given sequence. The triple-helix-forming oligonucleotide is preferably at least 15 nucleotides long, more preferably 25 nucleotides long, even more preferably 30 nucleotides or more long, and up to 50 or 100 nucleotides long.

[0593] The formation of a triple helix structure with target DNA induces steric and functional changes, blocking transcription initiation and elongation, enabling the introduction of desired sequence changes into endogenous DNA, and resulting in specific downregulation of gene expression. Examples of such gene expression repression in TFO-treated cells include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27: 1176-81, and Puri, et al, J Biol Chem, 2001;276:28991-98), downregulation of sequence-specific and target-specific expression of the Ets2 transcription factor, which is important in the pathogenesis of prostate cancer (Carbone, et al, Nucl Acid Res. 2003;31:833-43), and the pro-inflammatory ICAM-I gene (Besch et al, J Biol Chem, 2002;277:32473-79). Furthermore, Vuyisich and Beal recently demonstrated that sequence-specific TFOs bind to dsRNAs and inhibit the activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

[0594] Furthermore, TFOs designed according to the above principles can induce directed mutagenesis that can effectively perform DNA repair, thereby enabling both downregulation and upregulation of endogenous gene expression (Seidman and Glazer, J Clin Invest 2003; 112:487-94). Detailed descriptions of the design, synthesis, and administration of effective TFOs can be found in U.S. Patent Applications Nos. 2003 017068 and 2003 0096980 by Froehler et al., U.S. Patent Applications Nos. 2002 0128218 and 2002 0123476 by Emanuele et al., and U.S. Patent No. 5,721,138 by Lawn, the contents of which are incorporated herein by reference in their entirety.

[0595] Nucleic acid modification In some embodiments, the single-stranded oligonucleotide comprises at least one nucleic acid modification as described herein. For example, the at least one modification is selected from the group consisting of modified nucleoside linkages, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the single-stranded oligonucleotide. For example, the modification may be present in one of the RNA molecules.

[0596] 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 these nucleosides, including pentofuranosyl sugars, phosphate groups can be linked to the 2', 3', or 5' hydroxyl moieties of the sugar. In oligonucleotide formation, these phosphate groups covalently bond adjacent nucleosides to each other, forming linear polymer compounds. Within oligonucleotides, the phosphate groups are generally said to form the internucleoside backbone of the oligonucleotide. The naturally occurring bond or backbone of RNA and DNA is a 3'-to-5' phosphodiester bond.

[0597] 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 mimetic compounds known to those skilled in the art are suitable for the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared by these bases, or by synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine), and any one of the oligomeric modifications described herein. Alternatively, substitutions or modified analogs of any of the above bases and “universal bases” may be used. When a natural base is substituted with a non-natural and / or universal base, the nucleotide is said to contain a modified nucleobase and / or a nucleobase modification as described herein. Modified nucleobases and / or nucleobase modifications include natural, non-natural, and universal bases and contain a conjugated moiety, for example, a ligand as described herein. Preferred conjugated moieties for conjugation with a nucleobase contain a cationic amino group that can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0598] The oligomeric compounds described herein may also include nucleobase (often simply referred to as "bases" in the art) modification or substitution. The "unmodified" or "natural" nucleobases used herein include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularin, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, and 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 Nin, 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-(guanidinium alkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(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)pseudracil, 4-(thio)pseudracil, 2,4-(dithio)pseudracil, 5-(alkyl)pseudracil, 5-(methyl)pseudracil, 5-(alkyl)-2-(thio)pseudracil, 5-(methyl)-2-(thio)pseudracil, 5-(alkyl)-4-(thio)pseudracil, 5-(methyl)-4-(thio)pseudracil, 5-(alkyl)-2,4-(dithio)pseudracil Douracil, 5-(methyl)-2,4-(dithio)pseudracil, 1-substituted pseudouracil, 1-substituted 2(thio)pseudracil, 1-substituted 4-(thio)pseudracil, 1-substituted 2,4-(dithio)pseudracil, 1-(aminocarbonylethylenyl)pseudracil, 1-(aminocarbonylethylenyl)-2(thio)pseudracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudracil Douracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiadin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl,7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl,7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl,7-substituted 1,3-(diaza)-2-(oxo)-phenthiadin-1-yl,7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxadin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl, 7-(guanidinium alkylhydroxy )-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubralin, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimida Zolyl, Nitropyrazolyl, Nitrobenzimidazolyl, Nitroindazolyl, Aminoindolyl, Pyrrolopyrimidinyl, 3-(methyl)isocarbostyrillyl, 5-(methyl)isocarbostyrillyl, 3-(methyl)-7-(propynyl)isocarbostyrillyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, Imidizopyridinyl, 9-(methyl)-imidizopyridinyl, Pyrrolopyridinyl, Isocarbostyrillyl, 7-(propynyl)isocarbostyrillyl, Pripynyl-7-(aza) Indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenylon, tetracerenyl, pentaceryl, 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 - Substitution purine, N 6 - Substitution purine, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidine-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimi...

Claims

1. Formula (II) or Formula (III): (5'-Z １１ -3')-L-Q Ｓ -(5'-Z １２ -3') (II) (3’-Z １１ -5’)-L-Q Ｓ -(3’-Z １２ -5’) (III) [In the formula: Z １１ This is a first oligonucleotide containing 15 to 100 appropriately modified nucleotides that are substantially complementary to the target gene; Z １２ is a second oligonucleotide comprising 10 to 100 optionally modified nucleotides that are substantially complementary to Z １１ ; Z １１ and Z １２ It can form an intrachain double-strand region containing seven or more consecutive base pairs; Q Ｓ represents 0 to 12 appropriately modified nucleotides; L is any linking group. A single-stranded oligonucleotide shown, At least one nucleotide in formula (II) is a modified nucleotide, and At least one nucleotide in formula (III) is a modified nucleotide, In equation (II), Z １１ At least one nucleotide at the 3' end of, or of formula (III), Z １１ At least one nucleotide at the 5' end is L and Q in both cases. Ｓ Along with Z １１ and Z １２ Forms a loop region that connects Single-stranded oligonucleotide.

2. Z １１ The single-stranded oligonucleotide according to claim 1, wherein the oligonucleotide contains a loop at its 3' or 5' end.

3. Z １１ However, it includes W-LP, and W is Z １２ This allows for the formation of an intrachain double helix region of at least 7 base pairs, where LP, along with L as appropriate, has W and Z at its 3' or 5' end. １２ A single-stranded oligonucleotide according to claim 2, wherein a loop is formed between them.

4. W is Z １２ The single-stranded oligonucleotide according to claim 3, which can form an intrachain double-stranded region having base pairs with all of the nucleotides.

5. A single-stranded oligonucleotide is given by formula (IIa) or formula (IIIa): 【Chemistry 1】 [In the formula: Z １１ This includes W-LP, W is Z １２ This forms an intrachain double helix region of at least 7 base pairs, LP, along with L as appropriate, has W and Z at its 3' or 5' end. １２ Forming a loop between them, 【Chemistry 2】 This represents any existence of L, 【Transformation 3】 Q Ｓ Represents any existence of, 【Chemistry 4】 is, Z １１ Represents any overhang at the 5' or 3' end, and 【Transformation 5】 is, Z １２ [Represents any overhang at the 5' or 3' end] A single-stranded oligonucleotide according to claim 3, represented by [the specified formula].

6. Z １１ and Z １２ The single-stranded oligonucleotide according to claim 1 or 5, wherein each independently comprises 10 to 40 appropriately modified nucleotides.

7. Z １１ and Z １２ The single-stranded oligonucleotide according to claim 6, wherein each independently comprises 12 to 26 appropriately modified nucleotides.

8. Z １１ However, it contains 19 to 26 appropriately modified nucleotides, and Z １２ The single-stranded oligonucleotide according to claim 6, comprising 12 to 21 appropriately modified nucleotides.

9. L exists and the expression is #-(N) ｎ - ＊＊ [In the formula: # is Z １１ It is a coupling to, ＊＊ Q Ｓ or Z １２ It is a connection to, n is between 3 and 12; and Each N is independently a linked monomer having a chain length of three or more atoms. A single-stranded oligonucleotide according to claim 1 or 5, comprising a linking portion represented by .

10. A single-stranded oligonucleotide according to claim 9, wherein n is 3 to 8.

11. A single-stranded oligonucleotide according to claim 10, wherein n is 3.

12. The single-stranded oligonucleotide according to claim 9, wherein each N is independently a suitably modified nucleotide, Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, or Q368.

13. The single-stranded oligonucleotide according to claim 9, wherein L contains a triplet of Q304.

14. The position of L within the oligonucleotide is as follows: All linked monomers of L, together with LP, are W and Z. １２ Forming a loop between them; One or more linked monomers of L, together with LP, W and Z １２ A loop is formed between them, and one or more linked monomers of L are not in the loop region; One or more linked monomers of L, together with LP, W and Z １２ A loop is formed between them, and one or more linked monomers of L are not in the loop, Q Ｓ Being connected; and One or more linked monomers of L, together with LP, W and Z １２ A loop is formed between them, and one or more linked monomers of L are not in the loop, Z １２ Being connected A single-stranded oligonucleotide according to claim 9, characterized by one of the following.

15. Q Ｓ The single-stranded oligonucleotide according to claim 1 or 5, wherein the oligonucleotide is 1 to 6 appropriately modified nucleotides.

16. Q Ｓ The single-stranded oligonucleotide according to claim 15, wherein the oligonucleotide is two appropriately modified nucleotides.

17. Q Ｓ One or more nucleotides of Z １１ The single-stranded oligonucleotide according to claim 15, which forms a mismatch base pair with the nucleotide on the opposite side.

18. Q Ｓ However, these are two appropriately modified nucleotides, as follows: Q Ｓ Both nucleotides are Z １１ To form mismatch base pairs with the nucleotides on the opposite side, Z １２ Q is a nucleotide adjacent to it. Ｓ One of its nucleotides is Z １１ Forming a mismatch base pair with the opposite nucleotide in, or Q Ｓ Both nucleotides are Z １１ To form a base pair with the nucleotide on the opposite side of them, A single-stranded oligonucleotide according to claim 15, characterized by one of the following.

19. Z at non-loop ends １１ and Z １２ The single-stranded oligonucleotide according to claim 1 or 5, wherein the double-stranded region formed by has a blunt end.

20. Z １１ The single-stranded oligonucleotide according to claim 1 or 5, wherein the non-loop end has an overhang of 1 to 3 nucleotides in length.

21. Z １１ and Z １２ The single-stranded oligonucleotide according to claim 1 or 5, wherein the double-stranded region formed by contains three or fewer mismatched base pairs.

22. Z １１ The single-stranded oligonucleotide according to claim 1 or 5, wherein it contains three or fewer mismatches with respect to the target gene.

23. Z １１ All nucleotides in are modified nucleotides, and / or Z １２ The single-stranded oligonucleotide according to claim 1 or 5, wherein all nucleotides in the oligonucleotide are modified nucleotides.

24. A single-stranded oligonucleotide according to claim 1 or 5, further comprising one or more ligands (e.g., targeting ligands).

25. At least one ligand, via a linker or carrier as appropriate, Z １１ or Z １２ A single-stranded oligonucleotide according to claim 24, which is conjugated at an internal position.

26. At least one ligand, via a linker or carrier as appropriate, Z １１ or Z １２ The single-stranded oligonucleotide according to claim 24, which is conjugated to the 3' or 5' end of the oligonucleotide.

27. The single-stranded oligonucleotide according to claim 24, wherein at least one of the ligands comprises a lipophilic moiety.

28. The lipophilic portion is saturated or unsaturated C ４ ~C ３０ (For example, C ４ ~C １８ The single-stranded oligonucleotide according to claim 27, comprising a hydrocarbon chain and any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

29. The lipophilic portion, via a linker or carrier as appropriate, Z １１ or Z １２ A single-stranded oligonucleotide according to claim 28, conjugated at one or more internal positions of the same.

30. The internal position is Z １１ The 11th, 12th, and 13th positions from the 5' end are excluded and Z １１ Q paired at positions 11, 12, and 13 from the 5' end Ｓ and / or Z １２ The position of is excluded; for equation (II) or (IIa), Z １２ The 3' end and Z １１ Two or three terminal positions from the 5' end are appropriately excluded, and for equation (III) or (IIIa), Z １２ The 5' end and Z １１ The single-stranded oligonucleotide according to claim 29, wherein two or three terminal positions are appropriately excluded from the 3' end.

31. The single-stranded oligonucleotide according to claim 24, wherein at least one of the ligands is a targeting ligand selected from the group consisting of antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL ligands, and HDL ligands.

32. The single-stranded oligonucleotide according to claim 31, wherein at least one of the ligands is a carbohydrate-based ligand.

33. The single-stranded oligonucleotide according to claim 32, wherein the carbohydrate-based ligand is one or more GalNAc derivatives joined by a divalent or trivalent branched linker.

34. Carbohydrate-based ligands 【Transformation 6】 The single-stranded oligonucleotide according to claim 33.

35. Carbohydrate-based ligands, Z １１ Or Z １２ The 3' end, or Z １１ Or Z １２ A single-stranded oligonucleotide according to claim 32, which is conjugated at an internal position.

36. A single-stranded oligonucleotide according to claim 1 or 5, wherein the 5' terminal nucleotide includes a 5' phosphate modification or a 5' phosphate mimetic modification.

37. The single-stranded oligonucleotide according to claim 1 or 5, wherein the 5' or 3' terminal nucleotide includes a 2'-5'-linked nucleotide modification; or the 5' or 3' terminal nucleotide is conjugated to a debasalized nucleotide, an inverted nucleotide, or an inverted debasalized nucleotide (e.g., ribonucleotide) via a phosphodiester linkage, phosphorothioate linkage, or phosphodithioate linkage, as appropriate.

38. A single-stranded oligonucleotide according to claim 1 or 5, wherein the 5' or 3' terminal nucleotide is modified and comprises a linking portion containing mono-, di-, tri-, tetra-, penta-, or polyprolinol, or mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinol.

39. L, Q Ｓ or Z １２ connected to Z １１ The single-stranded oligonucleotide according to claim 1 or 5, wherein 3 to 5 terminal nucleotides of Z contain a modification selected from the group consisting of a 2'-deoxynucleotide (dN), a 2'-deoxy-2'-fluoronucleotide (fN), a ribonucleotide (rN), a 2'-O-methylnucleotide (mN), and a 2'-ara nucleotide (aN).

40. L, Q Ｓ , or Z １２ Connected, Z １１ The three terminal nucleotides are #-fN ＊＊ 、 #-dN-dN-dN- ＊＊ 、 #-dN-dN-rN- ＊＊ 、 #-dN-rN-dN- ＊＊ 、 #-rN-dN-dN- ＊＊ 、 #-rN-rN-dN- ＊＊ 、 #-rN-dN-rN- ＊＊ 、 #-dN-rN-rN- ＊＊ , and #-rN-rN-rN- ＊＊ [In the formula: # is Z １１ It is a coupling to, ＊＊ L, Q Ｓ , or Z １２ It is a coupling to dN represents a 2'-deoxyribonucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents ribonucleotide, mN represents 2'-O-methylnucleotide. A single-stranded oligonucleotide according to claim 39, having a modification selected from the group consisting of the following.

41. L, Q Ｓ , or Z １２ Connected, Z １１ The five terminal nucleotides are #-$N-$N-f ＊＊ 、 #-dN-dN-rN-dN-dN- ＊＊ 、 #-dN-dN-rN-rN-rN- ＊＊ 、 #-dN-dN-dN-dN-dN- ＊＊ 、 #-mN-mN-f ＊＊ 、 #-mN-mN-dN-dN-dN- ＊＊ 、 #-mN-mN-rN-dN-dN- ＊＊ , and #-mN-mN-rN-rN-rN- ＊＊ 、 [In the formula: # is Z １１ It is a coupling to, ＊＊ L, Q Ｓ , or Z １２ It is a coupling to dN represents a 2'-deoxyribonucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents ribonucleotide, mN represents 2'-O-methylnucleotide. A single-stranded oligonucleotide according to claim 39, having a modification selected from the group consisting of the following.

42. Z １１ However, Z １１ A single-stranded oligonucleotide according to claim 1 or 5, comprising at least one motif of three 2'-O-methyl modifications at the 11, 12, and 13 positions from the 5' end.

43. Z １２ However, Q as appropriate Ｓ The single-stranded oligonucleotide according to claim 1 or 5, further comprising at least one motif of three 2'-F modifications, wherein the nucleotide following the motif is not 2'-F modified.

44. Z １２ However, Q as appropriate Ｓ Along with that, its position is Z １１ The single-stranded oligonucleotide according to claim 43, which contains a 2'-O-methyl or 2'-F modification at a position two positions before the motif (n-2 position if the motif starts at position n), unless it is part of the motif.

45. The position of the motif in the oligonucleotide is as follows: The motif is Q Ｓ And Z １２ It is in the 1st and 2nd positions, and appropriately Z １１ It is 19 nucleotides long; The motif is Z １２ It is located in positions 1, 2 and 3, and appropriately Z １１ It is 20 nucleotides long; The motif is Z １２ It is located in positions 2, 3 and 4, and appropriately Z １１ It is 21 nucleotides long; The motif is Z １２ It is located in the 3rd, 4th, and 5th positions, and appropriately Z １１ It is 22 nucleotides long; or The motif is Z １２ It is located in positions 4, 5 and 6, and appropriately Z １１ It is 23 nucleotides long; A single-stranded oligonucleotide according to claim 43, characterized by one of the following.

46. The following nucleotide ligation modifications: (i) One or more internucleotide junctions within the six 3' terminal nucleotides are modified internucleotide junctions; and (ii) One or more nucleotide junctions within the six 5' terminal nucleotides are modified nucleotide junctions. A single-stranded oligonucleotide according to claim 1 or 5, further comprising one or more of the above.

47. The following nucleotide ligation modifications: (i) Two consecutive nucleotide junctions within the six 3' terminal nucleotides are modified nucleotide junctions; and (ii) The two consecutive nucleotide links within each of the six 5' terminal nucleotides must be modified nucleotide links. A single-stranded oligonucleotide according to claim 1 or 5, further comprising one or more of the above.

48. The following nucleotide ligation modifications: (i) Z of equation (II) １１ One or more internucleotide links within the eight 3' terminal nucleotides, or Z of formula (III) １１ One or more internucleotide junctions within the eight 5' terminal nucleotides are modified internucleotide junctions; (ii) Z of equation (II) １１ One or more internucleotide links within the six 5' terminal nucleotides, or Z of formula (III) １１ One or more internucleotide junctions within the six 3' terminal nucleotides are modified internucleotide junctions; (iii) Z of formula (II) or (IIa) １２ One or more internucleotide links within the six 5' terminal nucleotides, or Z of formula (III) or (IIIa). １２ One or more internucleotide junctions within the six 3' terminal nucleotides are modified internucleotide junctions; and (iv) Z of formula (II) or (IIa) １２ One or more internucleotide links within the six 3' terminal nucleotides, or Z of formula (III) or (IIIa). １２ One or more internucleotide junctions within the six 5' terminal nucleotides are modified internucleotide junctions. A single-stranded oligonucleotide according to claim 1 or 5, further comprising one of the above.

49. The following nucleotide ligation modifications: (i) Z of equation (II) or (IIa) １２ The linkage between two consecutive nucleotides within the three 5' terminal nucleotides, or the Z of formula (III) or (IIIa). １２ The two consecutive nucleotide links within the three 3' terminal nucleotides are modified nucleotide links; (ii) Z of equation (II) or (IIa) １２ The linkage between two consecutive nucleotides within the three 3' terminal nucleotides, or the Z of formula (III) or (IIIa). １２ The two consecutive nucleotide junctions within the three 5' terminal nucleotides are modified nucleotide junctions; and (iii) Z of formula (II) or (IIa) １１ A linkage between two consecutive nucleotides within three or four 5' terminal nucleotides, or Z of formula (III) or (IIIa). １１ The two consecutive nucleotide links within three or four 3' terminal nucleotides are modified nucleotide links. A single-stranded oligonucleotide according to claim 1 or 5, further comprising one or more of the above.

50. The single-stranded oligonucleotide according to any one of claims 46 to 49, wherein the modified nucleotide linkage is a phosphorothioate linkage.

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