Single-stranded loop oligonucleotide

Abstract

One aspect of the present invention relates to a single-stranded oligonucleotide having a sequence represented by formula (I): (5'-Z 1 -3')-Q 1 -L-Q 2 -(5'-Z 2 -3')(I). In formula (I), Z 1 is a first oligonucleotide comprising 15 to 100 optionally modified nucleotides that are substantially complementary to the target gene; Z 2 is a second oligonucleotide comprising 15 to 100 optionally modified nucleotides that are substantially complementary to Z 1 ; Z 1 and Z 2 can form an intrastrand double-stranded region containing 3 or more consecutive base pairs. L is a linking group. Q 1 and Q 2 each independently represent 0 to 12 optionally modified nucleotides. At least one nucleotide of formula (I) is a modified nucleotide. Other aspects of the present invention relate to pharmaceutical compositions and methods for inhibiting the expression of one or more target genes in a subject.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 364,715, filed May 13, 2022, and U.S. Provisional Patent Application No. 63 / 401,946, filed Aug. 29, 2022, both of which are hereby incorporated by reference in their entirety.

[0002] Array List This application contains an array list submitted in XML format, which is hereby incorporated by reference in its entirety. The XML copy created on May 12, 2023, is named 29520_1509 - PCT_ALN - 464_SL.xml and is 1,482,842 bytes in size.

[0003] The present invention generally relates to the field of RNA interference technology with single - stranded loop oligonucleotides.

Background Art

[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 pre - clinical and clinical development.

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

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, there is still a continuing need for an improved design of RNAi agents that simplifies the manufacturing and purification process while at the same time preserving or improving the effectiveness of the RNAi agent.

Means for Solving the Problems

[0007] One aspect of the present invention is of the 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 is 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 is 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 that can inhibit the expression of a target gene, wherein 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 2may each independently contain from 15 to 100 appropriately modified nucleotides. For example, Z 1 and Z 2 may each independently contain from 15 to 40, from 15 to 25, or from 19 to 23 appropriately modified nucleotides. In some embodiments, the first oligonucleotide Z 1 and the second oligonucleotide Z 2 may each independently contain at least 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. Z 1 and Z 2 may each independently have a length of from about 10 to about 50 nucleotides, from about 10 to about 40 nucleotides, from about 15 to about 40 nucleotides, from about 10 to about 35 nucleotides, from about 10 to about 30 nucleotides, from about 10 to about 25 nucleotides, from about 10 to about 20 nucleotides, from about 15 to about 50 nucleotides, from about 15 to about 40 nucleotides, from about 15 to about 35 nucleotides, from about 15 to about 30 nucleotides, from about 15 to about 25 nucleotides, from about 15 to about 20 nucleotides, from about 19 to about 23 nucleotides, from about 19 to about 21 nucleotides, or from about 18 to about 20 nucleotides. The nucleotides of the first oligonucleotide Z 1 and the second oligonucleotide Z 2 are each independently and may be appropriately modified. In some embodiments, Z 1 and Z 2 each contain the same number of appropriately modified nucleotides.

[0009] Q 1 and Q 2 may each independently contain from 0 to 12 appropriately modified nucleotides. For example, Q 1 and Q 2 may each independently contain from 0 to 10, from 0 to 6, from 0 to 4, from 0 to 3, from 0 to 2, from 1 to 6, from 1 to 4, from 1 to 3, or from 2 to 3 appropriately modified nucleotides. In some embodiments, Q 1and Q 2 are each 0. In some embodiments, Q 1 and Q 2 one of is 0. In some embodiments, Q 1 and Q 2 have the same number of appropriately modified nucleotides.

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

[0011] The first oligonucleotide Z 1 and the second oligonucleotide Z 2 can form an intermolecular double-stranded region containing 3 or more consecutive base pairs (e.g., a double-stranded region 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 region can contain 10-25, 15-25, 19-23, 19, 20, 21, 22, or 23 base pairs. Z 1 and Z 2 The intramolecular double-stranded region formed by can contain all consecutive base pairs or can contain 3 or fewer (e.g., 0, 1, 2, or 3) mismatched base pairs.

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

[0013] Chemical modifications to the nucleotide(s) can include internucleoside 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-methoxypropyl), 2'-O-alkyl (e.g., 2'-O-methyl), 2'-O-aryl, 2'-C-aryl, 2'-fluoro, 2'-deoxy, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (e.g., 2'-modified L-nucleosides, e.g., 2'-deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic and 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, including deoxynucleotide, 3'-terminal deoxythymidine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide (e.g., LNA), unlocked nucleotide (e.g., UNA), structurally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-aryl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxy-modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, unnatural base containing nucleotide, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing 5'-phosphorothioate group, nucleotide containing 5'-methylphosphonate group, nucleotide containing 5'-phosphate or 5'-phosphate mimetic, nucleotide containing vinylphosphonate, nucleotide containing glycol nucleic acid (GNA), nucleotide containing glycol nucleic acid (GNA) S isomer (S-GNA), nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide containing 2'-deoxythymidine-3'-phosphate, nucleotide containing 2'-deoxyguanosine-3'-phosphate, 2'-5'-linked nucleotide (“3'-RNA”), or a terminal nucleotide linked to a cholesteryl derivative or bisdecylamide group of dodecanoic acid.

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

[0017] In some embodiments, about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 1 is modified. In some embodiments, Z 2 about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 2 is modified. In some embodiments, all nucleotides of a single-stranded oligonucleotide of about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% are modified. For example, if 50% of all nucleotides are modified, 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 the single-stranded oligonucleotide are independently modified by 2'-O-methyl, 2'-O-aryl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropyl), 2'-deoxy, or 2'-fluoro.

[0019] In some embodiments, one or more of the internucleotide linkages of 5 out of 6 3'-terminal nucleotides are modified internucleotide linkages. In some embodiments, one or more of the internucleotide linkages of 5 out of 6 5'-terminal nucleotides are modified internucleotide linkages.

[0020] In some embodiments, one or more of the internucleotide linkages of 5 out of 6 5'-terminal nucleotides of Z 2 are modified internucleotide linkages. In some embodiments, one or more of the internucleotide linkages of 5 out of 6 5'-terminal nucleotides of Z 1 are modified internucleotide linkages.

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

[0022] In some embodiments, the single-stranded oligonucleotide further comprises a phosphate or 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 comprises 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 at the 5' end of Z 1 . 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'-vinyl phosphate.

[0023] In some embodiments, the 5' or 3' terminal nucleotide of the single-stranded oligonucleotide of formula (I) comprises a 2'-5'-linked nucleotide modification; or the 5' or 3' terminal nucleotide is conjugated to a abasic nucleotide, inverted nucleotide, or inverted abasic nucleotide (e.g., ribonucleotide) and may be via a phosphodiester, phosphorothioate, or phosphorodithioate bond.

[0024] In some embodiments, the 5' or 3' terminal nucleotide of the single-stranded oligonucleotide of formula (I) is modified and comprises 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 terminus, chiral modification (e.g., terminus, chiral phosphorus atom).

[0026] Site-specific, chiral modification to the internucleotide linkage can be present at the 5' terminus, 3' terminus, or both the 5' and 3' termini of the nucleotide sequence. This is referred to herein as "terminal, chiral" modification. The terminal modification can be present at the 3' or 5' terminal position of the terminal region, e.g., at the terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide sequence. Each of the chiral pure phosphorus atoms can be in either the Rp configuration or the Sp configuration, and combinations thereof. Details regarding chiral modification and chiral modified RNA agents can be found in WO2019 / 126651A1, which is hereby incorporated by reference in its entirety.

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

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

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

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

[0031] In some embodiments, each of Z 1 and Z 2 of a single-stranded oligonucleotide contains at least two consecutive phosphorothioate internucleotide linkage modifications. In one embodiment, each of Z 1 and Z 2 of a single-stranded oligonucleotide contains at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18-23 of the nucleotide sequence and at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1-5 of the nucleotide sequence, counted from the 5' end of the nucleotide sequence.

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

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

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

[0035] In some embodiments, the PN-linkage has the formula -N(R)P(=X)(OH)O- or -OP(=X)(OH)N(R)-, -O-P(NR)(=X)O-, N(SO 2 R)P(=X)(OH)O- or -OP(=X)(OH)N(SO 2 R)-, or -O-P(NSO 2 R)(=X)O- [wherein X is O or S; R can be an appropriately substituted alkyl, aryl, heteroaryl, or heterocyclyl; or NR can be an appropriately substituted cyclic guanidine moiety, an appropriately substituted triazolyl group, or a Tmg group (

[0036]

Chemical formula

[0037] In some embodiments, the PN linkage comprises a cyclic guanidine moiety that is optionally substituted. For example, the PN linkage

[0038]

Chemical formula

[0039] In some embodiments, the PN linkage comprises a triazole moiety (e.g., an optionally substituted triazole group). For example, the PN linkage

[0040]

Chemical formula

[0041] In some embodiments, the PN linkage comprises an alkyne moiety (e.g., an optionally substituted alkynyl group). For example, the PN linkage

[0042]

Chemical formula

[0043] In some embodiments, the PN linkage is a Tmg group (

[0044]

Chemical formula

[0045] [Chemical formula] [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.

[0046] More suitable PN linkages can include those described in WO2019 / 032642 and WO2021 / 030778, which are hereby incorporated by reference in their entirety.

[0047] In some embodiments, L in formula (I) is a cleavable linking group. In some embodiments, the cleavable linking group is cleavable in a homogenate of any type of cell, a tritosome, a cytosol, or an endosome. For example, the cleavable linking group can be cleavable in a liver homogenate, a liver tritosome, a liver lysosome, a liver cytosol, a liver endosome, a brain homogenate, a brain tritosome, a brain lysosome, a brain cytosol, or a brain endosome. In certain embodiments, the cleavable linking group is a redox-cleavable linker (e.g., a reductive-cleavable linker; e.g., a disulfide group), an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., an ester group), a peptidase-cleavable linker (e.g., an ester group), or an endosome-cleavable linker (or a protease-cleavable linker, e.g., a carbohydrate linker).

[0048] In some embodiments, the cleavable linking group (tether) is an endosome-cleavable linker or a protease-cleavable linker, such as a carbohydrate linker, and the 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).

[0049] In some embodiments, L of formula (I) contains a linking moiety represented by the formula: #-(N) n - ** wherein # is a bond to Q 1 and ** is a bond to Q 2 ; n is from 3 to 12; and each N is independently a linking moiety. For example, each N can independently be a linking monomer having a chain length of 3 or more atoms.

[0050] As used herein, the term "chain length" refers to the number of atoms in the shortest linear chain formed by the linking monomer. For example,[[]]

[0051]

Chemical formula

[0052]

Chemical formula

[0053]

Chemical formula

[0054]

Chemical formula

[0055]

Chemical formula

[0056] In some embodiments, one or more of the linking moieties (N) in L of formula (I) can be appropriately modified nucleotides.

[0057] In some embodiments, one or more of the linking moieties in L of formula (I) are independently selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methyl nucleotide (mN), and 2'-ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide). The ara-nucleotide has opposite stereochemical characteristics at the 2'-carbon atom compared to the ribonucleotide.

[0058] In certain embodiments, one or more linking moieties (N) in L of formula (I) can contain a modified internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester (which may contain a linking phosphorus atom in either the Rp or Sp configuration), hydrogen phosphonate, alkyl phosphonate or aryl phosphonate, phosphoramidate (which may contain a linking phosphorus atom in either the Rp or Sp configuration), phosphorothioate (which may contain a linking phosphorus atom in either the Rp or Sp configuration), methylene methylimino, nitrogen-modified phosphorus-containing linkage (PN linkage) (which may contain a linking phosphorus atom in either the Rp or Sp configuration), thiodiester, thiocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, borano phosphate, borano phosphate ester, amide, hydroxylamino, siloxane, dialkylsiloxane, carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal, formacetal, oxime, methyleneimino, methylenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether, thioacetamide, and combinations thereof.

[0059] In certain embodiments, one or more linking moieties (N) in L of formula (I) are: an aliphatic saturated or unsaturated alkyl chain; a phosphorus-containing linkage including phosphoric acid, phosphonate, phosphoramidate (which may include a linked phosphorus atom in either Rp or Sp configuration), phosphodiester, phosphotriester (which may include a linked phosphorus atom in either Rp or Sp configuration), phosphorothioate (which may include a linked phosphorus atom in either Rp or Sp configuration), and a nitrogen-modified phosphorus-containing linkage (PN linkage) (which may include a linked phosphorus atom in either Rp or Sp configuration); a (poly)ethylene glycol chain including diethylene glycol, triethylene glycol, tetra-, penta-, hexa-, hepta-, octa-, nona-, or decaethylene glycol; glycerol or a glycerol ester; an aminoalkyl ether; and moieties selected from the group consisting of combinations thereof.

[0060] In some embodiments, one or more linking moieties (N) in L of formula (I) may contain moieties selected from the group consisting of DNA, RNA, disulfide, amide, a functionalized monosaccharide or oligosaccharide of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0061] In some embodiments, one or more linking moieties (N) in L of formula (I) are independently

[0062]

Chemical formula

[0063] In some embodiments, one or more linking moieties (N) in L of formula (I) are independently

[0064]

Chemical formula

[0065] In some embodiments, one or more linking moieties (N) in L of formula (I) are mono, di, tri, tetra, penta, or polyprolinol optionally conjugated to a ligand; mono, di, tri, tetra, penta, or polyhydroxyprolinol optionally conjugated to a ligand; optionally modified nucleotides; or combinations thereof.

[0066] In some embodiments, L of formula (I) contains one or more mono, di, tri, tetra, penta or polyprolinol optionally conjugated to a ligand; and one or more optionally modified nucleotides.

[0067] In some embodiments, L of formula (I) contains one or more mono, di, tri, tetra, penta or polyhydroxyprolinol optionally conjugated to a ligand; and one or more optionally modified nucleotides.

[0068] In some embodiments, one or more linking moieties (N) in L of formula (I) are

[0069] [Table 1] TIFF2025516680000016.tif90160 and may include a portion selected from the group consisting of.

[0070] In some embodiments, L of formula (I) is of the formula: #-(N) n - **contains a linking moiety represented by the formula: In this formula, # is a bond to Q 1 is a bond to ** is a bond to Q 2 ; n is from 3 to 12; each N is independently a suitably 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.

[0071] In some embodiments, L in formula (I) contains from 3 to 5 2'-deoxynucleotides, triplets of 2'-deoxy-2'-fluoronucleotides, triplets of ribonucleotides, triplets of 2'-O-methylnucleotides, or triplets of Q304.

[0072] In some embodiments, L in formula (I) is as follows: #-mN-mN-mN-mN-mN- ** , #-rN-rN-rN-rN-rN- ** , #-rN-rN-fN-fN-fN- ** , #-dN-dN-fN-fN-fN- ** , #-dN-rN-rN-rN-dN- ** , #-dN-dN-dN-dN-dN- ** , #-mN-mN-dN-dN-dN- ** , #-mN-mN-rN-dN-dN- ** , #-mN-mN-rN-rN-rN- ** , and #-mN-mN-fN-fN-fN- ** , [wherein: dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents a ribonucleotide, mN represents a 2'-O-methyl nucleotide] and contains one of the following.

[0073] In some embodiments, L of 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--- ** , [wherein: dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents a ribonucleotide, mN represents a 2'-O-methyl nucleotide] and contains one of the following.

[0074] In the above embodiments, one or more internucleotide linkages between nucleotides in L can independently be a modified internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration), hydrogen phosphonate, alkyl phosphonate or aryl phosphonate, phosphoramidate (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration), and nitrogen-modified phosphorus-containing linkage (PN linkage) (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration).

[0075] In certain embodiments, L in formula (I) can contain one or more linking moieties selected from the group consisting of a triazole linkage, an amide linkage, a sulfide or disulfide linkage, a phosphate linkage, an oxime linkage, a hydrazo linkage, an N,N'-dialkylenehydrazo linkage, a methyleneimino linkage, a methylenecarbonylamino linkage, a methylenemethylimino linkage, a methylenehydrazo linkage, a methylenedimethylhydrazo linkage, a methyleneoxymethylimino linkage, a hydroxylamino linkage, a formacetal linkage, an alkyl or aryl linkage, a PEG linkage, an ether linkage, a thioether linkage, a thiodiester linkage, a thiocarbamate linkage, a thioacetamide linkage, a sulfonic acid linkage, a sulfonamide linkage, a sulfonic acid ester linkage, a thioformacetal linkage, a urea linkage, a carbonate linkage, an amine linkage, a maleimide-thioether linkage, a phosphodiester linkage, a phosphotriester linkage, a hydrogen phosphonate linkage, an alkyl or aryl phosphonic acid linkage, a phosphoramidate linkage, a phosphorothioate linkage, a nitrogen-modified phosphorus-containing linkage (PN-linkage), a phosphoroselenate linkage, a boranophosphate linkage, a boranophosphate ester linkage, a sulfonamide linkage, a carbamate linkage, a carboxamide linkage, a carboxymethyl linkage, a carboxylate ester linkage, a siloxane linkage, a dialkylsiloxane linkage, an ethylene oxide linkage, and combinations thereof.

[0076] In certain embodiments, L in formula (I) can 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, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0077] In some embodiments, L in formula (I) contains a nucleotide-based linker (tether). In some embodiments, L contains a non-nucleotide-based linker (tether).

[0078] 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.

[0079] In certain embodiments, the cleavable linking group (tether) is

[0080]

Chemical formula

[0081]

Chemical formula

[0082] In certain embodiments, the cleavable linking group (tether) is as follows: -(CH 2 ) 12 -(C 12 linker or Q50), -(CH 2 ) 6 -S-S-(CH 2 ) 6 -(C6-S-S-C6 linker or Q51), Q151(

[0083]

Chemical formula

[0084]

Chemical formula

[0085] In certain embodiments, the cleavable linker (tether) comprises a nucleic acid linker that is 1 to 15 nucleotides in length. For example, the nucleic acid linker can have a length of 2 to 7, 5 to 7, 2 to 5, or 3, 4, or 5 nucleotides of appropriate modification.

[0086] In certain embodiments, the cleavable linker (tether) comprises a nucleic acid linker comprising one or more nucleotides selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, deoxyribonucleotides, and ribonucleotides. In one embodiment, all nucleic acid linker nucleotides are of the same type of nucleotide. In one embodiment, the nucleic acid linker consists entirely of 2'-O-methyl nucleotides, consists entirely of 2'-fluoro nucleotides, or consists entirely of deoxyribonucleotides.

[0087] In certain embodiments, the cleavable linking group (tether) comprises a polynucleotide comprising a modified ribonucleotide sequence, a polynucleotide comprising one or more modifications selected from the group consisting of 2'-O-methyl ribonucleotide modification, 2'-fluoro-ribonucleotide modification, 2'-5'-linked nucleotides having different 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro), glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, hexanol nucleic acid (HNA) modification, abasic ribose modification, abasic deoxyribose modification, and abasic hydroxyproline modification.

[0088] In some embodiments, the linking group L of the single-stranded oligonucleotide of formula (I) comprises a nucleotide-based cleavable linking group (tether) that is cleavable by DICER. In some embodiments, the single-stranded oligonucleotide comprises a substrate cleavable by DICER.

[0089] In certain embodiments, the single-stranded oligonucleotide comprises a cleavable linking group (nucleotide-based or non-nucleotide-based) that can generate a 5'-monophosphate metabolite in at least one nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 1 and / or Z 2 ).

[0090] In some embodiments, the single-stranded oligonucleotide may further comprise one or more ligands (e.g., targeting ligands). In one embodiment, Z 1 comprises at least one ligand (e.g., targeting ligand) at the 5' or 3' end of the sequence. In one embodiment, Z 2 comprises at least one ligand (e.g., targeting ligand) at the 5' or 3' end of the sequence. In one embodiment, Z 1 and Z 2 each comprise at least one ligand (e.g., targeting ligand) at the 5' or 3' end of the sequence.

[0091] In certain embodiments, at least one ligand is a lipophilic moiety.

[0092] In one embodiment, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, deoxycholic acid (DCA), dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecyl glycerol, 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 certain embodiments, the lipid is a fatty acid (such as omega-3 fatty acids, for example) selected from the group consisting of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0093] In some embodiments, the lipophilic moiety is a saturated or unsaturated C 4 ~C 30 hydrocarbon chain (e.g., C 4 ~C 30 alkyl or alkenyl), and contains any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0094] In some embodiments, the lipophilic moiety is a saturated or unsaturated C 6 ~C 18 hydrocarbon chain (e.g., linear C 6 ~C 18 alkyl or alkenyl), for example, a saturated or unsaturated C 16 hydrocarbon chain (e.g., linear C 16 alkyl or alkenyl). In some embodiments, the lipophilic moiety is a saturated or unsaturated C 14 ~C 24 hydrocarbon chain (e.g., linear C 14 ~C 24(alkyl or alkenyl), for example, saturated or unsaturated C 22 hydrocarbon chain (e.g., linear C 22 (alkyl or alkenyl). For example, one or more non-terminal positions of a single-stranded oligonucleotide may have the following structure:

[0095] [Chemical formula] [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]. The modification shown in formula (1) is herein referred to as "2'-C 16 ". In another example, one or more non-terminal positions of a single-stranded oligonucleotide may have the following structure:

[0096] [Chemical formula] [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]. The modification shown in formula (2) is herein referred to as "2'-C 22 ".

[0097] Similar modifications that replace the n-hexadecyl chain or the n-docosanyl chain with a C 4 ~C 30 hydrocarbon chain are referred to as "2'-C 4 ~C 30 hydrocarbon chain" (or replacement with a C 6 ~C 18 hydrocarbon chain or a C 14 ~C 24 hydrocarbon chain is referred to as "2'-C 6 ~C 18 hydrocarbon chain" or "2'-C 14 ~C 24 hydrocarbon chain").

[0098] In related embodiments, Z 1 and Z 2 at least one of one or more non-terminal nucleotide positions is 2'-C of formula (1) 4 ~C 30 hydrocarbon chain structure, 2'-C 6 ~C 18 hydrocarbon chain structure, 2'-C 14 ~C 24 hydrocarbon chain structure, 2'-C 16 structure, or 2'-C of formula (2) 22 has a structure.

[0099] In one embodiment, Z 1 and Z 2 one or more non-terminal nucleotide positions of both are 2'-C of formula (1) 4 ~C 30 hydrocarbon chain structure, 2'-C 6 ~C 18 hydrocarbon chain structure, 2'-C 14 ~C 24 hydrocarbon chain structure, 2'-C16 structure, or 2'-C22 structure of formula (2).

[0100] In some embodiments, the lipophilic moiety contains one or more phospholipids.

[0101] In some embodiments, the lipophilic moiety contains one or more lipids or lipophilic ligands disclosed in International PCT Application Publication Nos. WO2019 / 232255A1 and WO2021 / 108662A1, and U.S. Patent No. 10,184,124, which are hereby incorporated by reference in their entirety.

[0102] In some embodiments, the ligand has the following formula:

[0103] [Chemical formula] [wherein, in "C10-TEG-", n is 1, and in "C16-TEG-", n is 7] includes one or more of.

[0104] In some embodiments, the ligand includes all those disclosed in International PCT Application Publications WO2017 / 053999, WO2019 / 118916, WO2022 / 031433, WO2022 / 056269, WO2022 / 056273, and WO2022 / 056277, which are hereby incorporated by reference in their entirety.

[0105] In some embodiments, Z 1 and Z 2 at least one of which includes one or more internal positions (i.e., non-terminal positions) excluding positions 9 to 12 of the nucleotide sequence; for example, positions 4 to 8 and 13 to 18 of the nucleotide sequence, counted 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 conjugated independently to positions 4, 6, 7, and 8 of the nucleotide sequence.

[0106] In some embodiments, Z 1 and Z 2 at least one of which includes one or more lipophilic moieties conjugated independently to position 6 of the nucleotide sequence, counted from the 5' end of the nucleotide sequence. In one embodiment, Z 1 and Z 2 each includes a lipophilic moiety conjugated to position 6 of the nucleotide sequence; the lipophilic moiety may include a saturated or unsaturated C 6 -C 18 hydrocarbon chain, or may include a saturated or unsaturated C 14 -C 24 hydrocarbon chain; the lipophilic moiety may include a saturated or unsaturated C 16 hydrocarbon chain or a saturated or unsaturated C 22 hydrocarbon chain.

[0107] In some embodiments, Z 1 and Z 2At least one of them comprises 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, and positions 15 and 17 of the nucleotide sequence, respectively counted from the 5' end of the nucleotide sequence as position 1, and one or more lipophilic moieties conjugated independently thereto.

[0108] In some embodiments, at least one ligand is a targeting ligand selected from the group consisting of an antibody, an antigen, folic acid, a receptor ligand, a hydrocarbon, an aptamer, an integrin receptor ligand, a chemokine receptor ligand, transferrin, biotin, a serotonin receptor ligand, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In one embodiment, at least one ligand is an integrin receptor ligand.

[0109] The targeting ligand can be conjugated to an internal position of the nucleotide sequence (e.g., Z 11 and Z 12 ) via a linker or carrier as appropriate. Alternatively, the targeting ligand can be conjugated to the 3' end or 5' end of Z 11 and Z 12 via a linker or carrier as appropriate.

[0110] In certain embodiments, at least one ligand is a hydrocarbon-based ligand. The hydrocarbon-based ligand can 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, a glycosylated polyamino acid, or a lectin.

[0111] In certain embodiments, the hydrocarbon-based ligand is an ASGPR ligand. For example, the ASGPR ligand is a divalent or trivalent branched linker, such as:

[0112] [Chemistry] One or more GalNAc derivatives conjugated by

[0113] In certain embodiments, at least one ligand can be conjugated to the 3'-end, 5'-end, or internal position of a nucleotide sequence (e.g., Z 1 and Z 2 ).

[0114] In some embodiments, at least one ligand can be conjugated to a single-stranded oligonucleotide via direct conjugation to the ribosugar of the oligonucleotide. Alternatively, the ligand can be conjugated to the single-stranded oligonucleotide via one or more linkers (tethers) and / or a carrier.

[0115] In some embodiments, the ligand can be conjugated to a single-stranded oligonucleotide via a monovalent or branched divalent or trivalent linker.

[0116] In some embodiments, the ligand can be conjugated to a single-stranded oligonucleotide via a carrier that replaces one or more nucleotides (plural available). The carrier can be a cyclic group or an 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, pyridazinonyl, tetrahydrofuranyl, and decalinyl. In one embodiment, the acyclic group is a moiety based on a serinol skeleton or a diethanolamine skeleton.

[0117] In some embodiments, the single-stranded oligonucleotide is (a) Z1 and Z 2 each independently contains from 19 to 23 optionally modified nucleotides; (b) Q 1 and Q 2 each independently contains from 0 to 2 optionally modified nucleotides; (c) Z 1 and Z 2 the double-stranded region formed by them contains 3 or fewer mismatched base pairs; (d) Z 1 and Z 2 the double-stranded region formed by them forms blunt ends; (e) at least one nucleotide of Z 2 is a modified nucleotide; (f) at least one nucleotide of Z 1 is a modified nucleotide; (g) Z 2 contains at least one modified internucleotide linkage; (h) Z 1 contains at least one modified internucleotide linkage; (i) the 5'-terminal nucleotide contains a 5'-phosphate or 5'-phosphate mimetic modification; (j) the 3'-terminal nucleotide is conjugated to a ligand and may be via a linker; (k) Z 1 contains 3 or fewer mismatches to the target gene; (l) Z 1 and Z 2 each independently contains from 19 to 23 optionally modified nucleotides; and (m) L has the formula: #-(N) n - **[wherein n is 3 to 5; each N is independently a nucleotide optionally modified, Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, or Q368], and contains a linking moiety represented by] can be characterized by one or more of.

[0118] In some embodiments, the single-stranded oligonucleotide can be characterized by two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or all of the above features.

[0119] In some embodiments, the single-stranded oligonucleotide is (a) Z 1 and Z 2 each independently contain 21 nucleotides optionally modified; (b) Q 1 and Q 2 each independently contain 2 nucleotides optionally modified; (c) The double-stranded region formed by Z 1 and Z 2 contains 3 or fewer mismatched base pairs; (d) The double-stranded region formed by Z 1 and Z 2 forms blunt ends; (e) All nucleotides of Z 2 are modified nucleotides; (f) All nucleotides of Z 1 are modified nucleotides; (g) Z 2 contains at least one internucleotide linkage between two consecutive modified nucleotides; (h) Z 1 contains at least one internucleotide linkage between two consecutive modified nucleotides; (i) Z 1 The 5'-terminal nucleotide of which contains a 5'-phosphate or 5'-phosphate mimetic modification; (j) Z 2 The 3'-terminal nucleotide of which is conjugated to a ligand and may be via a linker; (k) Z 1 contains three or fewer mismatches to the target gene; and (l) L is of the formula: #-(N) n - ** [wherein n is 5; each N is independently a suitably modified nucleotide or Q304] and contains a linking moiety represented by and may be characterized by one or more of.

[0120] In some embodiments, the single-stranded oligonucleotide may be characterized by two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or all of the above features.

[0121] Another aspect of the invention is of 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), [wherein: Z 11 is a first oligonucleotide comprising 15 to 100 suitably modified nucleotides that is substantially complementary to the target gene; Z 12 is a second oligonucleotide comprising 10 to 100 suitably modified nucleotides that is substantially complementary to Z 11 ; and Z 11 and Z12 can form an intrastrand double-stranded region containing 7 or more consecutive base pairs; Q S represents a nucleotide modified as appropriate from 0 to 12, L is an arbitrary linking group is a single-stranded oligonucleotide represented by at least one nucleotide of formula (II) is a modified nucleotide; and at least one nucleotide of formula (III) is a modified nucleotide, where for formula (II), at least one nucleotide at the 3'-end of Z 11 , and for formula (III), at least one nucleotide at the 5'-end of Z 11 together with L and Q S forms a loop region connecting Z 11 and Z 12 relates to a single-stranded oligonucleotide. Regarding single-stranded oligonucleotides.

[0122] In some embodiments, the single-stranded oligonucleotide can be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, a microRNA mimic, a supermir, an aptamer, a U1 adapter, a triple-strand-forming oligonucleotide, an RNA activator, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a heteroduplex-forming oligonucleotide, or a single-stranded siRNA (ss-siRNA) oligonucleotide.

[0123] The first oligonucleotide Z 11 and the second oligonucleotide Z 12 can each independently contain from 10 to 100 appropriately modified nucleotides. For example, Z 11 and Z 12each independently contains 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 independently may contain at least 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. Z 11 and Z 12 each independently may have a length of about 10 - about 50 nucleotides, about 10 - about 40 nucleotides, about 10 - about 35 nucleotides, about 10 - about 30 nucleotides, about 10 - about 26 nucleotides, about 10 - about 23 nucleotides, about 10 - about 21 nucleotides, about 12 - about 50 nucleotides, about 12 - about 40 nucleotides, about 12 - about 35 nucleotides, about 12 - about 30 nucleotides, about 12 - about 26 nucleotides, about 12 - about 23 nucleotides, about 12 - about 21 nucleotides, about 15 - about 50 nucleotides, about 15 - about 40 nucleotides, about 15 - about 35 nucleotides, about 15 - about 30 nucleotides, about 15 - about 26 nucleotides, about 15 - about 23 nucleotides, about 15 - about 21 nucleotides, about 19 - about 50 nucleotides, about 19 - about 40 nucleotides, about 19 - about 35 nucleotides, about 19 - about 30 nucleotides, about 19 - about 26 nucleotides, about 19 - about 23 nucleotides, about 19 - about 21 nucleotides, or about 18 - about 20 nucleotides.

[0124] In some embodiments, Z 11 and Z 12 each independently contains appropriately modified nucleotides of 10 - 40. In some embodiments, Z 11 and Z 12 each independently contains appropriately modified nucleotides of 12 - 26.

[0125] In some embodiments, Z 11 and Z 12 each contain the same number of optionally modified nucleotides. In some embodiments, Z 11 contains more optionally modified nucleotides than Z 12 . In some embodiments, Z 11 contains 19 to 26 optionally modified nucleotides, and Z 12 contains 12 to 21 optionally modified nucleotides.

[0126] In some embodiments, the single-stranded oligonucleotide can be cleaved by a linking group L. The first oligonucleotide Z 11 is cleaved into an antisense strand that is substantially complementary to a target gene (e.g., target mRNA or DNA), and the second oligonucleotide Z 12 can be cleaved into a sense strand that is substantially complementary to Z 11 .

[0127] Q S can contain 0 to 12 optionally modified nucleotides. For example, Q S can contain 0 to 10, 0 to 6, 0 to 4, 0 to 3, 0 to 2, 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3 optionally modified nucleotides. In some embodiments, Q S is 0. In some embodiments, Q S is 1 to 6 optionally modified nucleotides. In some embodiments, Q S is 2 optionally modified nucleotides. In some embodiments, Q S is 1 optionally modified nucleotide.

[0128] In some embodiments, one or more nucleotides of Q S form a mismatched base pair with the opposite nucleotides of Z 11 . In some embodiments, Q S is 2 optionally modified nucleotides, as follows: Q S Both nucleotides of are the opposite of Z11 forming mismatched base pairs with those opposite nucleotides thereof Q S one nucleotide of forms mismatched base pairs with the opposite nucleotide of Z 11 forming mismatched base pairs with the opposite nucleotide of Z (e.g., Q 12 next to Z S the nucleotide of forms mismatched base pairs with those opposite nucleotides of Z 11 (), or Q S both nucleotides of form base pairs with those opposite nucleotides of Z 11 characterized by one of is characterized by one of

[0129] The first oligonucleotide Z 11 is substantially complementary to the target gene, i.e., Z 11 contains 3 or fewer (e.g., 0, 1, 2, or 3) mismatches with the target gene. In some embodiments, the target gene can be mRNA, pre-mRNA, microRNA, pre-miRNA, long non-coding RNA (lncRNA), or DNA.

[0130] The first oligonucleotide Z 11 and the second oligonucleotide Z 12 can form an intrastrand duplex region, e.g., containing 7 or more consecutive base pairs. In some embodiments, Z 11 and Z 12 can form an intrastrand duplex 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 can form an intrastrand duplex region having base pairs with all nucleotides of Z 12 . Z 11 and Z 12The intrastrand double-stranded region formed by can contain all consecutive base pairs or can contain a maximum of 3 mismatched base pairs (e.g., 0, 1, 2, or 3). In some embodiments, Z 11 and Z 12 The intrastrand double-stranded region formed by contains 1 mismatched base pair.

[0131] In some embodiments, the first oligonucleotide Z 11 and the second oligonucleotide Z 12 can form an intrastrand double-stranded region in the seed region of Z 11 (e.g., the seed region of the antisense strand; e.g., positions 2 to 8 at the 5' end of the antisense strand).

[0132] In some embodiments, the first oligonucleotide Z 11 contains a loop at the 3' end or the 5' end. In some embodiments, the first oligonucleotide Z 11 is W-LP [where W can form an intrastrand double-stranded region of at least 7 base pairs with Z 12 and LP, together with L as appropriate, forms a loop between W and Z 12 at the 3' end or the 5' end]. In some embodiments, W and Z 12 can form an intrastrand double-stranded 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 can form an intrastrand double-stranded region having base pairs with all nucleotides of Z 12 . The intrastrand double-stranded region formed by W and Z 12 can contain all consecutive base pairs or can contain 3 or fewer (e.g., 0, 1, 2, or 3) mismatched base pairs.

[0133] In some embodiments, the single-stranded oligonucleotide has the formula (IIa) or formula (IIIa):

[0134]

Chemical formula

[0135]

Chemical formula

[0136]

Chemical formula

[0137]

Chemical formula

[0138]

Chemical formula

[0139] In some embodiments, the double-stranded region formed by Z 11 and Z 12 at the non-loop ends has blunt ends. In some embodiments, W and Z 12 ​The double-stranded region formed thereby has blunt ends.

[0140] In some embodiments, Z at the non-loop end 11 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,

[0141]

Chemical formula

[0142] In some embodiments, Z 12 has an overhang of 1 to 3 nucleotides in length. In some embodiments,

[0143]

Chemical formula

[0144] In some embodiments, the overhang is 1 nucleotide in length. In some embodiments, the overhang is 2 nucleotides in length. In some embodiments, the overhang is 3 nucleotides in length.

[0145] Each of the nucleotides within the single-stranded oligonucleotide can be independently and optionally modified. Each of the nucleotides within the first oligonucleotide Z 11 and the second oligonucleotide Z 12 can be independently and optionally modified.

[0146] In some embodiments, the single-stranded oligonucleotide comprises at least one chemical modification. In some embodiments, the first oligonucleotide Z 11 and the second oligonucleotide Z 12Each of them contains at least one chemical modification. In some embodiments, W contains 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 nucleotides of are modified nucleotides. In some embodiments, all nucleotides of a single-stranded oligonucleotide are modified.

[0147] Chemical modifications to the nucleotide(s) can include internucleoside linkage modifications, nucleobase modifications, sugar modifications, or combinations thereof.

[0148] 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-methoxypropyl), 2'-O-alkyl, 2'-O-aryl, 2'-C-aryl, 2'-fluoro, 2'-deoxy, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (e.g., 2'-modified L-nucleosides, e.g., 2'-deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic and open-chain alkyls, and combinations thereof.

[0149] In certain embodiments, the chemical modification is selected from the group consisting of at least one modified nucleotide, deoxynucleotide, 3'-terminal deoxythymidine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide (LNA), unlocked nucleotide (UNA), structurally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-aryl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxy-modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, unnatural base containing nucleotide, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing 5'-phosphorothioate group, nucleotide containing 5'-methylphosphonate group, nucleotide containing 5'-phosphate or 5'-phosphate mimetic, nucleotide containing vinylphosphonate, nucleotide containing glycol nucleic acid (GNA), nucleotide containing glycol nucleic acid (GNA) S isomer (S-GNA), nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide containing 2'-deoxythymidine-3'-phosphate, nucleotide containing 2'-deoxyguanosine-3'-phosphate, 2'-5'-linked nucleotide (“3'-RNA”), or a terminal nucleotide linked to a cholesteryl derivative or a dodecanonic acid bisdecylamide group.

[0150] In certain embodiments, the chemical modification is a 2'-modification selected from the group consisting of 2'-O-methyl, 2'-O-aryl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropyl), 2'-deoxy, 2'-fluoro, and combinations thereof.

[0151] In some embodiments, about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 11 is modified. In some embodiments, about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 12 is 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, when 50% of all nucleotides are modified, 50% of all nucleotides present in the single-stranded oligonucleotide contain at least one modification described herein.

[0152] In one embodiment, at least 50% of the nucleotides of the single-stranded oligonucleotide are independently modified by 2'-O-methyl, 2'-O-aryl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropyl), 2'-deoxy, or 2'-fluoro.

[0153] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one or more of the following internucleotide linkage modifications; (i) One or more internucleotide linkages within the six 3'-terminal nucleotides are modified internucleotide linkages; and (ii) One or more internucleotide linkages within the six 5'-terminal nucleotides are modified internucleotide linkages.

[0154] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) is Z of formula (II) 11further comprises one or more internucleotide linkages within one of the eight 3'-terminal nucleotides, or Z of formula (III) 11 one or more internucleotide linkages within the eight 5'-terminal nucleotides of 11 are modified internucleotide linkages.

[0155] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one or more of the following internucleotide linkage modifications; (i) The internucleotide linkage between two consecutive nucleotides within the six 3'-terminal nucleotides is a modified internucleotide linkage; and (ii) The internucleotide linkage between two consecutive nucleotides within the six 5'-terminal nucleotides is a modified internucleotide linkage.

[0156] In some embodiments, if the single-stranded oligonucleotide contains a terminal conjugation of a ligand to a 5'- or 3'-terminal nucleotide, or a terminal conjugation of an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide, then, at that terminus, the above-described internucleotide linkage modification to the terminal nucleotide can be removed.

[0157] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one of the following internucleotide linkage modifications; (i) One or more internucleotide linkages within the eight 3'-terminal nucleotides of Z of formula (II) (or IIa) 11 or one or more internucleotide linkages within the eight 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) are modified internucleotide linkages; 11 (ii) One or more internucleotide linkages within the six 5'-terminal nucleotides of Z of formula (II) (or IIa) 11 or one or more internucleotide linkages within the six 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) 11 ​One or more internucleotide linkages within one of the six 3'-terminal nucleotides are modified internucleotide linkages; (iii) Z of formula (II) (or (IIa)) 12 One or more internucleotide linkages within one of the six 5'-terminal nucleotides, or Z of formula (III) (or (IIIa)) 12 One or more internucleotide linkages within one of the six 3'-terminal nucleotides are modified internucleotide linkages; and (iv) Z of formula (II) (or (IIa)) 12 One or more internucleotide linkages within one of the six 3'-terminal nucleotides, or Z of formula (III) (or (IIIa)) 12 One or more internucleotide linkages within one of the six 5'-terminal nucleotides are modified internucleotide linkages.

[0158] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises one or more of the following internucleotide linkage modifications; (i) The internucleotide linkage between two consecutive nucleotides within three 5'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three 3'-terminal nucleotides of Z of formula (III) (or (IIIa)) is a modified internucleotide linkage; 12 The internucleotide linkage between two consecutive nucleotides within three 5'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three 3'-terminal nucleotides of Z of formula (III) (or (IIIa)) is a modified internucleotide linkage; 12 (ii) The internucleotide linkage between two consecutive nucleotides within three 3'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) is a modified internucleotide linkage; and (ii) The internucleotide linkage between two consecutive nucleotides within three 3'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) is a modified internucleotide linkage; and 12 The internucleotide linkage between two consecutive nucleotides within three 3'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) is a modified internucleotide linkage; and 12 (iii) The internucleotide linkage between two consecutive nucleotides within three or four 5'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three or four 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) is a modified internucleotide linkage; (iii) The internucleotide linkage between two consecutive nucleotides within three or four 5'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three or four 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) is a modified internucleotide linkage; 11 The internucleotide linkage between two consecutive nucleotides within three or four 5'-terminal nucleotides of Z of formula (II) (or (IIa)), or the internucleotide linkage between two consecutive nucleotides within three or four 5'-terminal nucleotides of Z of formula (III) (or (IIIa)) 11The linkage between two consecutive nucleotides within three or four 3'-terminal nucleotides is a modified inter-nucleotide linkage.

[0159] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) has one or more modified inter-nucleotide linkages between the 5'-terminal nucleotide of Z 12 and the first nucleotide of Q S In some embodiments, the single-stranded oligonucleotide of formula (III) (or (IIIa)) further comprises one or more modified inter-nucleotide linkages between the 3'-terminal nucleotide of Z 12 and the first nucleotide of Q S In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or IIIa) further comprises one or more modified inter-nucleotide linkages between the nucleotides of Q S

[0160] In some embodiments, in all of the above embodiments, the modified inter-nucleotide linkage is a phosphorothioate linkage.

[0161] In some embodiments, the single-stranded oligonucleotide of formula (II) (or (IIa)) or formula (III) (or (IIIa)) comprises at least two consecutive phosphorothioate or methylphosphonate inter-nucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least two blocks of two consecutive phosphorothioate or methylphosphonate inter-nucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least three blocks of two consecutive phosphorothioate or methylphosphonate inter-nucleotide linkage modifications.

[0162] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) has a nucleotide sequence (e.g., Z 11 and / or Z 12 ​) has at least two phosphorothioate internucleotide linkages within the first 6 nucleotides.

[0163] In some embodiments, the nucleotide sequence of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) comprises two blocks of phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphodiester internucleotide linkages.

[0164] In one embodiment, the nucleotide sequence of a single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) (e.g., Z 11 and / or Z 12 ) comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18 - 23 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence. In one embodiment, the nucleotide sequence of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1 - 5 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence.

[0165] In some embodiments, each of Z 11 and Z 12 of a single-stranded oligonucleotide comprises at least two consecutive phosphorothioate internucleotide linkage modifications. In one embodiment, each of Z 11 and Z 12 of a single-stranded oligonucleotide comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18 - 23 of the nucleotide sequence and at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1 - 5 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence.

[0166] In some embodiments, Z at the non-loop end 11 has an overhang that is 1 to 3 nucleotides in length. In one embodiment, Z at the non-loop end 11 has an overhang that is 2 nucleotides in length (e.g., the 3' end of Z 11 ) and has a phosphorothioate internucleotide linkage between the two overhangs. In one embodiment, Z at the non-loop end 11 has an overhang that is 2 nucleotides in length and has two phosphorothioate internucleotide linkages between the terminal 3 nucleotides (e.g., the 3' end of Z 11 ), two of the 3 nucleotides are overhang nucleotides, and the third is the complementary nucleotide next to the overhang nucleotide.

[0167] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises a phosphate or phosphate mimetic at the 5' end of the nucleotide sequence (e.g., Z 11 and / or Z 12 ). In some embodiments, the single-stranded oligonucleotide comprises a phosphate mimetic at the 5' end of the nucleotide sequence (e.g., Z 11 and / or Z 12 ). In one embodiment, at least one phosphate mimetic is at the 5' end of Z 11 . 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.

[0168] In some embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) further comprises at least one terminal chiral phosphorus atom.

[0169] In some embodiments, the 5'- or 3'-terminal nucleotide of the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) comprises a 2'-5'-linked nucleotide modification, or the 5'- or 3'-terminal nucleotide is conjugated to an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide (e.g., a ribonucleotide), and may be via a phosphodiester, phosphorothioate, or phosphodithioate linkage.

[0170] In some embodiments, the 5'- or 3'-terminal nucleotide of the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)) is modified and comprises a linking moiety containing mono-, di-, tri-, tetra-, penta- or polyprolinol, or mono-, di-, tri-, tetra-, penta- or polyhydroxyprolinol.

[0171] In some embodiments, at least one, two, three, four, or five of the terminal phosphorus-containing linkages at the 5'- or 3'-end of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) are not phosphorothioate linkages. In one embodiment, each of at least one, two, three, four, or five of the terminal phosphorus-containing linkages at the 5'- or 3'-end of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) is, independently of each other, a native phosphate group or a phosphodiester linkage, or a PN-linkage.

[0172] In some embodiments, at least one, two, three, four, or five of the terminal phosphorus-containing linkages at the 5'- or 3'-end of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) are each -N(R)P(=X)(OH)O- or -OP(=X)(OH)N(R)-, -O-P(NR)(=X)O-, N(SO 2R)P(=X)(OH)O- or -OP(=X)(OH)N(SO 2 R)-, or -O-P(NSO 2 R)(=X)O-[wherein X is O or S; R can be optionally substituted alkyl, aryl, heteroaryl or heterocyclyl; or NR can be an optionally substituted cyclic guanidine moiety, an optionally substituted triazolyl group, or a Tmg group

[0173]

Chemical formula

[0174] In some embodiments, each of at least one, two, three, four, or five terminal phosphorus-containing linkages at the 5' end or 3' end of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) is a PN linkage comprising an optionally substituted cyclic guanidine moiety, e.g.,

[0175]

Chemical formula

[0176] In some embodiments, each of at least one, two, three, four, or five terminal phosphorus-containing linkages at the 5' end or 3' end of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) is a PN linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group), e.g.,

[0177]

Chemical formula

[0178] In some embodiments, at least one, two, three, four, or five terminal phosphorus-containing linkages at the 5'-end or 3'-end of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) each are a PN linkage comprising an alkyne moiety (e.g., an optionally substituted alkynyl group), e.g.,

[0179]

Chemical formula

[0180] In some embodiments, at least one, two, three, four, or five terminal phosphorus-containing linkages at the 5'-end or 3'-end of a single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) each are a Tmg group

[0181]

Chemical formula

[0182]

Chemical formula

[0183] In all of the above embodiments, the PN linkage can be stereochemically controlled.

[0184] 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 to Z11 Three to five of the terminal nucleotides contain a modification selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-arabonucleotide (aN).

[0185] In some embodiments, in the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), L, Q S , or Z 12 connected to Z 11 has three terminal nucleotides that 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- ** [wherein: # is a bond to Z 11 , ** is a bond to L, Q S , or Z 12 and dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents a ribonucleotide, mN represents a 2'-O-methylnucleotide] and has a modification selected from the group consisting of.

[0186] In some embodiments, in the single-stranded oligonucleotide of formula (II) (or IIa) or formula (III) (or (IIIa)), L, Q S , or Z 12 connected to Z 11 The five terminal nucleotides of 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 [wherein: # is a bond to Z 11 , ** is a bond to L, Q S , or Z 12 is a bond to dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents a ribonucleotide, mN represents a 2'-O-methyl nucleotide] has a modification selected from the group consisting of.

[0187] In some embodiments, the first oligonucleotide Z 11 contains at least one motif of three consecutive 2'-O-methyl modifications at positions 11, 12, and 13 from the 5' end of Z 11 , and the nucleotide following the motif is not 2'-O-methyl modified.

[0188] In some embodiments, the second oligonucleotide Z 12 optionally contains, together with Q S at least one motif of three consecutive 2'-F modifications, and the nucleotide following the motif is not 2'-F modified.

[0189] In some embodiments, the positions of the motifs of three consecutive modifications (three consecutive 2'-O-methyl modifications or three consecutive 2'-F modifications) are as follows: The motif is at positions 1 and 2 of Q S and Z 12 and Z 11 may be 19 nucleotides in length; The motif is at positions 1, 2, and 3 of Z 12 and Z 11 may be 20 nucleotides in length; The motif is at positions 2, 3, and 4 of Z 12 and Z 11 may be 21 nucleotides in length; The motif is at positions 3, 4, and 5 of Z 12 and Z 11 may be 22 nucleotides in length; or The motif is at positions 4, 5, and 6 of Z 12 and Z 11 may be 23 nucleotides in length; and is characterized by one of them.

[0190] In some embodiments, Z 12 optionally contains, together with Q S a 2'-O-methyl or 2'-F modification at a position that is before the second position of the motif (position n - 2 if the motif is at position n), unless the position is part of Z 11

[0191] In some embodiments, L is a cleavable linking group. In some embodiments, the cleavable linking group is cleavable in a homogenate of any type of cell, a Tritosome, a cytosol, or an endosome. For example, the cleavable linking group can be cleavable in a liver homogenate, a liver Tritosome, a liver lysosome, a liver cytosol, a liver endosome, a brain homogenate, a brain Tritosome, a brain lysosome, a brain cytosol, or a brain endosome. In certain embodiments, the cleavable linking group is a redox-cleavable linker (e.g., a reductive-cleavable linker; e.g., a disulfide group), an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., an ester group), a peptidase-cleavable linker (e.g., an ester group), or an endosome-cleavable linker (or a protease-cleavable linker, e.g., a carbohydrate linker).

[0192] In some embodiments, the cleavable linking group (tether) is an endosome-cleavable linker or a protease-cleavable linker, e.g., a carbohydrate linker, and the linker is cleaved at least 1.25 times faster in a cell (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).

[0193] 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 a bond to Z 11 and ** is Q S or Z 12a linkage thereto; n is from 3 to 12; each N is, independently, a linking monomer having a chain length of 3 or more atoms. For example, each N can independently be a linking monomer having a chain length of 3 or more atoms. "Chain length" is defined hereinabove. 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 3.

[0194] In some embodiments, one or more of the linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) can be appropriately modified nucleotides.

[0195] In some embodiments, one or more of the linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) are independently selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methyl nucleotide (mN), and 2'-ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide).

[0196] In certain embodiments, one or more linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) may contain a modified internucleotide linkage selected from the group consisting of phosphodiester, phosphorotriester (which may contain a linking phosphorus atom in either the Rp or Sp configuration), hydrogen phosphonate, alkyl phosphonate or aryl phosphonate, phosphoramidate (which may contain a linking phosphorus atom in either the Rp or Sp configuration), phosphorothioate (which may contain a linking phosphorus atom in either the Rp or Sp configuration), methylene methylimino, nitrogen-modified phosphorus-containing linkage (PN linkage) (which may contain a linking phosphorus atom in either the Rp or Sp configuration), thiodiester, thiocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, boranophosphate, boranophosphate ester, amide, hydroxylamino, siloxane, dialkylsiloxane, carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal, formacetal, oxime, methyleneimino, methylenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether, thioacetamide, and combinations thereof.

[0197] In certain embodiments, one or more linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) can contain a moiety selected from the group consisting of an aliphatic saturated or unsaturated alkyl chain; a phosphorus-containing linkage including phosphoric acid, phosphonate, phosphoramidate (which may contain a linked phosphorus atom in either the Rp or Sp configuration), phosphodiester, phosphotriester (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 a nitrogen-modified phosphorus-containing linkage (PN linkage) (which may contain a linked phosphorus atom in either the Rp or Sp configuration); a (poly)ethylene glycol chain including diethylene glycol, triethylene glycol, tetra-, penta-, hexa-, hepta-, octa-, nona-, or decaethylene glycol; glycerol or a glycerol ester; an aminoalkyl ether; and combinations thereof.

[0198] In some embodiments, one or more linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) can contain a moiety selected from the group consisting of DNA, RNA, disulfide, amide, an oligosaccharide of a functionalized monosaccharide or galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0199] In some embodiments, one or more linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) are independently

[0200] [Chemical formula] [wherein: Base is an appropriately modified nucleobase, R D is C 4 ~ 30 alkyl, C 4 ~ 30 alkenyl, or C 4 ~ 30is alkynyl] may be selected from the group consisting of.

[0201] In some embodiments, one or more linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) are mono-, di-, tri-, tetra-, penta-, or polyprolinols optionally conjugated with a ligand; mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinols optionally conjugated with a ligand; optionally modified nucleotides; or combinations thereof.

[0202] In some embodiments, L of formula (II) (or IIa) or formula III (or IIIa) contains one or more mono-, di-, tri-, tetra-, penta- or polyprolinols optionally conjugated with a ligand; and one or more optionally modified nucleotides.

[0203] In some embodiments, L of formula (II) (or IIa) or formula III (or IIIa) contains one or more mono-, di-, tri-, tetra-, penta- or polyhydroxyprolinols optionally conjugated with a ligand; and one or more optionally modified nucleotides.

[0204] In some embodiments, one or more linking moieties (N) in L of formula (II) (or IIa) or formula III (or IIIa) may independently be 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.

[0205] In some embodiments, each linking moiety (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.

[0206] In some embodiments, L of formula (II) (or IIa) or formula III (or IIIa) contains three to five 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.

[0207] In some embodiments, the position of L in formula (II) (or IIa) or formula III (or IIIa) is as follows: All linking monomers of L, together with LP, form a loop between W and Z 12 ; One or more linking monomers of L, together with LP, form a loop between W and Z 12 and one or more linking monomers of L are not in the loop region; One or more linking monomers of L, together with LP, form a loop between W and Z 12 and one or more linking monomers of L are not in the loop but are connected to Q S ; and One or more linking monomers of L, together with LP, form a loop between W and Z 12 and one or more linking monomers of L are not in the loop but are connected to Z 12 ; characterized by one of the following.

[0208] In the above embodiments, one or more internucleotide linkages in L of formula (II) (or IIa) or formula III (or IIIa) can independently be a modified internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration), hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration), and nitrogen-modified phosphorus-containing linkages (PN linkages) (which may contain a linking phosphorus atom in either Rp configuration or Sp configuration).

[0209] In certain embodiments, L of formula (II) (or IIa) or formula III (or IIIa) can contain one or more linking moieties selected from the group consisting of triazole linkages, amide linkages, sulfide or disulfide linkages, phosphate linkages, oxime linkages, hydrazo linkages, N,N'-dialkylenehydrazo linkages, methyleneimino linkages, methylenecarbonylamino linkages, methylenemethylimino linkages, methylenehydrazo linkages, methylenedimethylhydrazo linkages, methyleneoxymethylimino linkages, hydroxylamino linkages, formacetal linkages, alkyl or aryl linkages, PEG linkages, ether linkages, thioether linkages, thiodiester linkages, thiocarbamate linkages, thioacetamide linkages, sulfonic acid linkages, sulfonamide linkages, sulfonic acid ester linkages, thioformacetal linkages, urea linkages, carbonate linkages, amine linkages, maleimide-thioether linkages, phosphodiester linkages, phosphotriester linkages, hydrogen phosphonate linkages, alkyl or aryl phosphonate linkages, phosphoramidate 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.

[0210] In certain embodiments, L of 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, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0211] In some embodiments, L of 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).

[0212] 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.

[0213] In certain embodiments, the cleavable linker (tether) contains a moiety of formula (CL-1) or (CL-2) as described above.

[0214] In certain embodiments, the cleavable linker (tether) is as follows: -(CH 2 ) 12 -(C12 linker or Q50), -(CH 2 ) 6 -S-S-(CH 2 ) 6 -(C6-S-S-C6 linker or Q51), Q151, Q173, -CH 2 CH 2 O-(CH 2 CH 2 ) n -CH 2 CH 2 O-CH 2 CH 2 O - [wherein, n is 0 or 1 to 20]; -(CH 2 ) 9 -(CH 2 ) n -CH 2 - [wherein, n is 0 or 1 to 20]; Mono-, di-, tri-, tetra-, penta- or polyprolinol optionally conjugated with a ligand, Mono-, di-, tri-, tetra-, penta- or polyhydroxyprolinol optionally conjugated with a ligand comprising a moiety selected from.

[0215] In certain embodiments, the cleavable linker (tether) comprises a nucleic acid linker that is 1 to 15 nucleotides in length. For example, the nucleic acid linker can be of a length of 2 to 7, 5 to 7, 2 to 5, or 3, 4, or 5 nucleotides, optionally modified.

[0216] In certain embodiments, the cleavable linker (tether) comprises a nucleic acid linker comprising one or more nucleotides selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, deoxyribonucleotides, and ribonucleotides. In one embodiment, all nucleic acid linker nucleotides are of the same type of nucleotide. In one embodiment, the nucleic acid linker completely comprises 2'-O-methyl nucleotides, completely comprises 2'-fluoro nucleotides, or completely comprises deoxyribonucleotides.

[0217] In certain embodiments, the cleavable linker (tether) comprises a polynucleotide comprising a modified ribonucleotide sequence and may be a polynucleotide comprising one or more modifications selected from the group consisting of 2'-O-methyl ribonucleotide modifications, 2'-fluoro-ribonucleotide modifications, 2'-5'-linked nucleotides having different 3'-modifications (3'-ribo, 3'-O-methyl, 3'-deoxy, 3'-fluoro), glycol nucleic acid (GNA) modifications, locked nucleic acid (LNA) modifications, hexanol nucleic acid (HNA) modifications, abasic ribose modifications, abasic deoxyribose modifications, and abasic hydroxyprolinol modifications.

[0218] In some embodiments, the linker L of the single-stranded oligonucleotide of formula (II) (or IIa) or formula III (or IIIa) comprises a nucleotide-based cleavable linker (tether) that is cleavable by DICER. In some embodiments, the single-stranded oligonucleotide comprises a substrate cleavable by DICER.

[0219] In certain embodiments, the single-stranded oligonucleotide of formula (II) (or IIa) or formula III (or IIIa) has at least one nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 11 and / or Z 12 ) that contains a cleavable linker (nucleotide-based or non-nucleotide-based) that can generate a 5'-monophosphate metabolite.

[0220] 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 comprises at least one ligand (e.g., targeting ligand) at the 5' or 3' end of the sequence. In one embodiment, Z 12 comprises at least one ligand (e.g., targeting ligand) at the 5' or 3' end of the sequence. In one embodiment, Z11 and Z 12 Each of 12 contains at least one ligand (e.g., a targeting ligand) at the 5' or 3' end of the array.

[0221] In some embodiments, at least one ligand is conjugated to an internal position of a nucleotide sequence (e.g., Z 11 and Z 12 ) via an optional linker or carrier. In some embodiments, at least one ligand is conjugated to the 3' or 5' end of Z 11 or Z 12 via an optional linker or carrier. In some embodiments, at least one ligand can be conjugated to a single-stranded oligonucleotide via direct conjugation to the ribosugar of the oligonucleotide. Alternatively, the ligand can be conjugated to a single-stranded oligonucleotide via one or more linkers (tethers), and / or a carrier.

[0222] In some embodiments, the internal position can refer to one upstream or downstream of positions 1 to 4 nucleotides of Q S . In some embodiments, the internal position can refer to one upstream or downstream of positions 1 to 4 nucleotides of the nucleotides of Z 11 that pair with positions 11, 12, and 13 from the 5' end of Z 12 .

[0223] Z 12 For the purpose of counting internal positions for conjugation of only the ligand, the terminal nucleotide (in 12 of Z S ) connected to Q 12 can be considered an internal position.

[0224] In some embodiments, the internal position is from which the nucleotides of Q S and / or Z 12 directly connected to the loop region are excluded; and / or Z11 excluding the 2nd or 14th position from the 5'-end; and / or Z 11 excluding the 11th, 12th, and 13th positions from the 5'-end; and / or Z 11 Q paired to the 11th, 12th, and 13th positions from the 5'-end of S and / or Z 12 excluding the position; and / or In formula (II) or (IIa), Z 12 excluding two or three terminal positions from the 3'-end of Z and the 5'-end of Z 11 ; and / or In formula (III) or (IIIa), Z 12 excluding two or three terminal positions from the 5'-end of Z and the 3'-end of Z 11 can be characterized by .

[0225] In some embodiments, the ligand can be conjugated to a single-stranded oligonucleotide via a monovalent or branched divalent or trivalent linker.

[0226] In some embodiments, the ligand can be conjugated to a single-stranded oligonucleotide via a carrier that replaces one or more nucleotides (s). The carrier can be a cyclic group or an 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, pyridazinonyl, tetrahydrofuranyl, and decalinyl. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0227] In certain embodiments, at least one ligand comprises a lipophilic moiety.

[0228] In one embodiment, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, docosanoic 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 certain embodiments, the lipid is a fatty acid (such as an omega-3 fatty acid) selected from the group consisting of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0229] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C 4 ~C 30 hydrocarbon chain (e.g., C 4 ~C 30 alkyl or alkenyl), and any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0230] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C 6 ~C 18 hydrocarbon chain (e.g., linear C 6 ~C 18 alkyl or alkenyl), e.g., a saturated or unsaturated C 16 hydrocarbon chain (e.g., linear C 16 alkyl or alkenyl). In some embodiments, the lipophilic moiety contains a saturated or unsaturated C 14 ~C 24 hydrocarbon chain (e.g., linear C 14 ~C 24 alkyl or alkenyl), e.g., a saturated or unsaturated C 22 hydrocarbon chain (e.g., linear C 22It contains (alkyl or alkenyl). For example, one or more non-terminal positions of a single-stranded oligonucleotide are, as described above herein, of formula (I) [wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives), and the n-hexadecyl chain is the lipophilic moiety] "2'-C 16 " modification. In another example, one or more non-terminal positions of a single-stranded oligonucleotide are, as described above herein, of formula (2) [wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives), and the n-docosanyl chain is the lipophilic moiety] "2'-C 22 " modification.

[0231] Replacing the n-hexadecyl chain or the n-docosanyl chain with a C 4 ~C 30 hydrocarbon chain for a similar modification is referred to as "2'-C 4 ~C 30 hydrocarbon chain" (or the substitution with a C 6 ~C 18 hydrocarbon chain or a C 14 ~C 24 hydrocarbon chain is referred to as "2'-C 6 ~C 18 hydrocarbon chain" or "2'-C 14 ~C 24 hydrocarbon chain").

[0232] In related embodiments, at least one of one or more non-terminal nucleotide positions of Z 11 and Z 12 has a 2'-C 4 ~C 30 hydrocarbon chain structure, 2'-C 6 ~C 18 hydrocarbon chain structure, 2'-C 14 ~C 24 hydrocarbon chain structure, 2'-C 16 structure, or a 2'-C 22 structure of formula (2).

[0233] In some embodiments, the lipophilic moiety contains one or more phospholipids.

[0234] In some embodiments, the lipophilic moiety contains one or more lipids or lipophilic ligands disclosed in International PCT Application Publication Nos. WO2019 / 232255A1 and WO2021 / 108662A1, and U.S. Patent No. 10,184,124, which are hereby incorporated by reference in their entireties.

[0235] In some embodiments, the ligand includes one or more ligands of formula (L-1), (L-2), (L-3), or (L-4) as described above herein.

[0236] In some embodiments, the ligand includes those disclosed in International PCT Application Publication Nos. WO2017 / 053999, WO2019 / 118916, WO2022 / 031433, WO2022 / 056269, WO2022 / 056273, and WO2022 / 056277, which are hereby incorporated by reference in their entireties.

[0237] In some embodiments, the lipophilic moiety is saturated or unsaturated C 4 ~C 30 (e.g., C 4 ~C 18 ) hydrocarbon chain and contains any functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0238] In some embodiments, the lipophilic moiety is conjugated to one or more internal positions of Z 11 or Z 12 via a linker or carrier as appropriate.

[0239] In some embodiments, Z 11 and Z 12At least one of them comprises one or more internal positions (i.e., non-terminal positions) excluding positions 9 to 12 of the nucleotide sequence; for example, counting from the 5' end of the nucleotide sequence as position 1, positions 4 to 8 and 13 to 18 of the nucleotide sequence; positions 5, 6, 7, 15, and 17 of the nucleotide sequence; or one or more lipophilic moieties conjugated independently to positions 4, 6, 7, and 8 of the nucleotide sequence.

[0240] In some embodiments, Z 11 and Z 12 At least one of them comprises one or more lipophilic moieties conjugated independently to position 6 of the nucleotide sequence, counting from the 5' end of the nucleotide sequence. In one embodiment, Z 11 and Z 12 each comprises a lipophilic moiety conjugated to position 6 of the nucleotide sequence; the lipophilic moiety may comprise a saturated or unsaturated C 4 ~C 30 (e.g., C 4 ~C 18 ) hydrocarbon chain, or may comprise a saturated or unsaturated C 14 ~C 24 hydrocarbon chain; the lipophilic moiety may comprise a saturated or unsaturated C 16 hydrocarbon chain or a saturated or unsaturated C 22 hydrocarbon chain.

[0241] In some embodiments, Z 11 and Z 12 At least one of them comprises one or more non-terminal positions of the nucleotide sequence; for example, counting from the 5' end of the nucleotide sequence as position 1, positions 6 to 10 and 15 to 18 of the nucleotide sequence; one or more lipophilic moieties conjugated independently to positions 15 and 17 of the nucleotide sequence.

[0242] 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, Z11 the 2nd or 14th position from the 5'-end is excluded; and / or Z 11 the 11th, 12th, and 13th positions from the 5'-end are excluded; and / or Z 11 Q paired with the 11th, 12th, and 13th positions from the 5'-end of S and / or Z 12 the position is excluded; and / or in formula (II) or (IIa), Z 12 the 3'-end of and Z 11 two or three terminal positions from the 5'-end may be excluded; and / or in formula (III) or (IIIa), Z 12 the 5'-end of and Z 11 two or three terminal positions from the 3'-end may be excluded.

[0243] In some embodiments, at least one ligand is a targeting ligand selected from the group consisting of an antibody, an antigen, folic acid, a receptor ligand, a hydrocarbon, an aptamer, an integrin receptor ligand, a chemokine receptor ligand, transferrin, biotin, a serotonin receptor ligand, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In one embodiment, at least one ligand is an integrin receptor ligand.

[0244] The targeting ligand can be conjugated to an internal position of a nucleotide sequence (e.g., Z 11 and Z 12 ) via a linker or a carrier as appropriate. Alternatively, the targeting ligand can be conjugated to the 3'-end or 5'-end of Z 11 or Z 12 via a linker or a carrier as appropriate.

[0245] In certain embodiments, at least one ligand is a hydrocarbon-based ligand. Hydrocarbon-based ligands can 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 polyamino acid, or lectin.

[0246] In some embodiments, the hydrocarbon-based ligand is a divalent or trivalent branched linker, such as:

[0247]

Chemical formula

[0248] In some embodiments, one or more targeting ligands (e.g., hydrocarbon-based ligands) are conjugated at an internal position of Z 11 excluding positions 2 or 14.

[0249] In the above aspect of the present invention regarding 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) can be 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl. In one embodiment, the phosphate mimetic is 5'-vinylphosphonate (VP). 5'-VP can be either the 5'-E-VP isomer (i.e., trans-vinylphosphonate), the 5'-Z-VP isomer (i.e., cis-vinylphosphate), or a mixture thereof.

[0250] 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.

[0251] In another embodiment, the single-stranded oligonucleotide further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand (i.e., Z 1 ). The phosphate mimetic may be 5'-vinylphosphonate (VP). When the phosphate mimetic is 5'-vinylphosphonate (VP), the 5'-terminal nucleotide has the following structure,

[0252]

Chemical formula

[0253] In one embodiment, R 5’ is =C(H)-P(O)(OH) 2 and the double bond between the C5’ carbon and R5’ is in the E orientation. In another embodiment, R is methoxy, and R 5’ is =C(H)-P(O)(OH) 2 and the double bond between the C5’ carbon and R5’ is in the E orientation. In another embodiment, X is S, R is methoxy, and R 5’ is =C(H)-P(O)(OH) 2 and the double bond between the C5’ carbon and R 5’ is in the E orientation.

[0254] In some embodiments, the -CH 2 OH group at the 4’ position of the 5’-terminal nucleotide is replaced with a phosphonate mimic of the formula -O-CH 2 -P(O)(OR) 2 [wherein each R is independently hydrogen or C 1~4 alkyl (e.g., one R group is hydrogen and one R group is methyl; or both R groups are hydrogen)].

[0255] In one embodiment, the phosphonate mimic is 5’-cyclopropylphosphonate (VP), i.e., the CH 2 OH group at the 4’ position of the 5’-terminal nucleotide is replaced with a group of the formula -Cy-P(O)(OR) 2 [wherein Cy is a cyclopropyl ring and each R is independently hydrogen or C 1~4 alkyl (e.g., one R group is hydrogen or both R groups are hydrogen)].

[0256] In some exemplary embodiments, the 5’-terminal phosphonate mimic is

[0257] [Chemical formula] or a salt thereof (e.g., sodium salt), wherein B may be a modified nucleobase (e.g., U).

[0258] In some embodiments, the 5'-end phosphate mimetic is part of a modified 5'-end nucleotide. For example, the phosphate mimetic has the structure

[0259] [Chemical formula] [wherein B may be a modified nucleobase] and can be part of a modified 5'-end nucleotide having the structure.

[0260] In some embodiments, the 5'-end phosphate mimetic may also include a 5'-phosphate prodrug or a 5'-phosphonate prodrug. In some embodiments, the 5'-phosphate prodrug or the 5'-phosphonate prodrug has a structure of a formula disclosed in WO2022 / 147214, which is incorporated herein by reference. In some exemplary embodiments, the 5'-phosphate prodrug or the 5'-phosphonate prodrug is Pmmds (

[0261] [Chemical formula] , ((4SR,5SR)-3,3,5-trimethyl-1,2-dithiolan-4-ol) phosphodiester); cPmmds (

[0262] [Chemical formula] , ((4SR,5RS)-3,3,5-trimethyl-1,2-dithiolan-4-ol) phosphodiester (Cis Pmmds)); PdArls (

[0263] [Chemical formula] ((4SR,5RS)-5-phenyl-3,3-dimethyl-1,2-dithiolan-4-ol) phosphodiester); PdAr3s(

[0264]

Chem.

[0265]

Chem.

[0266]

Chem.

[0267]

Chem.

[0268]

Chem.

[0269]

Chem.

[0270]

Chem.

[0271]

Chem.

[0272] [Chemical formula] , X is O / S), Pd / Pds(

[0273] [Chemical formula] , X is O / S) is as follows.

[0274] In some exemplary embodiments, the 5'-phosphate prodrug or 5'-phosphonate prodrug is

[0275] [Chemical formula] is as follows. An siRNA containing one of the above-listed 5'-modified phosphate prodrugs generally has activity comparable to that of an siRNA containing 5'-VP. In some exemplary embodiments, the 5'-phosphate prodrug or 5'-phosphonate prodrug is

[0276] [Chemical formula] An siRNA containing one of the above-listed 5'-modified phosphate prodrugs generally has improved stability compared to that of an siRNA containing 5'-VP and has activity better than or comparable to that of an siRNA containing 5'-VP.

[0277] 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 linked.

[0278] Another aspect of the present invention relates to an oligonucleotide construct comprising two single-stranded oligonucleotides of formula (II) (or IIa) or (III) (or IIIa), wherein the two single-stranded oligonucleotides are covalently linked.

[0279] 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).

[0280] In some embodiments, the covalent linkage between the two single-stranded oligonucleotides is present at the linking group L of each single-stranded oligonucleotide.

[0281] In some embodiments, the two single-stranded oligonucleotides are covalently linked by a tethering group selected from the group consisting of oxime, aminooxy, triazole or fused triazole, phosphodiester, phosphotriester, hydrogen phosphonate, alkyl phosphonate or aryl phosphonate, phosphoramidate, phosphorothioate, nitrogen-modified phosphorus-containing linkage (PN linkage), methylene methylimino, thiodiester, thiocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, boranophosphate, boranophosphate ester, amide, hydroxylamino, siloxane, dialkylsiloxane, carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal, formacetal, methyleneimino, methylenecarbonylamino, methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether, thioacetamide, and combinations thereof.

[0282] In some embodiments, the tethering group is an oxime, an aminooxy, or a triazole or fused triazole. Exemplary processes for covalently linking an exemplary tethering group and two single-stranded oligonucleotides to form an oligonucleotide construct are shown in Schemes 7.1 - 7.4 below.

[0283] The two single-stranded oligonucleotides may be the same or different.

[0284] In some embodiments, the two single-stranded oligonucleotides forming the oligonucleotide construct are the same. In one embodiment, the single-stranded oligonucleotide forming the oligonucleotide construct is derived from formula (I). In one embodiment, the single-stranded oligonucleotide forming the oligonucleotide construct is derived from formula (II) (or IIa). In one embodiment, the single-stranded oligonucleotide forming the oligonucleotide construct is derived from formula (III) (or IIIa).

[0285] In some embodiments, the two single-stranded oligonucleotides forming the oligonucleotide construct are different.

[0286] 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, the Z 1 and / or Z 2 of one single-stranded oligonucleotide contains a modification different from the Z 1 and / or Z 2 of the other single-stranded oligonucleotide. In some embodiments, the L of one single-stranded oligonucleotide is different from the L of the other single-stranded oligonucleotide. In some embodiments, the Q 1 and / or Q 2is different from Q of other single-stranded oligonucleotides 1 and / or Q 2 is different. In some embodiments, one single-stranded oligonucleotide contains a ligand different from that of another single-stranded oligonucleotide. For example, one single-stranded oligonucleotide contains a ligand, while another single-stranded oligonucleotide does not contain a ligand or contains a different ligand. In some embodiments, one single-stranded oligonucleotide contains a ligand at a position different from the ligand on another single-stranded oligonucleotide.

[0287] 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, the Z of one single-stranded oligonucleotide 11 and / or Z 12 contains a modification different from the Z of another single-stranded oligonucleotide 11 and / or Z 12 In some embodiments, the L of one single-stranded oligonucleotide is different from the L of another single-stranded oligonucleotide. For example, one single-stranded oligonucleotide contains L, while another single-stranded oligonucleotide does not contain L or contains a different L. In some embodiments, the Q of one single-stranded oligonucleotide S is different from the Q of another single-stranded oligonucleotide S For example, one single-stranded oligonucleotide contains Q S while another single-stranded oligonucleotide does not contain Q S or contains a different Q SIt contains. In some embodiments, one single-stranded oligonucleotide contains a ligand different from that of another single-stranded oligonucleotide. For example, one single-stranded oligonucleotide contains a ligand, while another single-stranded oligonucleotide does not contain a ligand or contains a different ligand. In some embodiments, one single-stranded oligonucleotide contains a ligand at a position different from that of the ligand on another single-stranded oligonucleotide.

[0288] In some embodiments, the two single-stranded oligonucleotides forming the oligonucleotide construct are two different single-stranded oligonucleotides having one single-stranded oligonucleotide derived from formula (I) and another single-stranded oligonucleotide derived from formula (II) (or IIa) or formula (III) (or IIIa).

[0289] Another aspect of the present invention relates to a pharmaceutical composition comprising the above single-stranded oligonucleotide according to formula (I) and a pharmaceutically acceptable excipient.

[0290] Another aspect of the present invention relates to a pharmaceutical composition comprising the above single-stranded oligonucleotide according to formula (II) (or (IIa)) or formula (III) (or (IIIa)) and a pharmaceutically acceptable excipient.

[0291] Another aspect of the present invention relates to a pharmaceutical composition comprising the above oligonucleotide construct containing two single-stranded oligonucleotides according to formula (I), (II) (or (IIa)), or formula (III) (or (IIIa)) and a pharmaceutically acceptable excipient.

[0292] All of the above embodiments related to single-stranded oligonucleotides, nucleotide sequence(s), all variables defined in formula (I) and formula (I), all variables defined in formula (II)-(IIa) and formula (II)-(IIa), all variables defined in formula (III)-(IIIa) and formula (III)-(IIIa), chemical modifications on the nucleotide sequence, the linking group L within the oligonucleotide, the tethering group covalently linking two single-stranded oligonucleotides, and the ligands and ligand conjugations disclosed in the above aspects of the present invention related to single-stranded oligonucleotides are suitable for this aspect of the present invention regarding pharmaceutical compositions.

[0293] Another aspect of the present invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising contacting the single-stranded oligonucleotide as described above with the cells of the subject by 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.

[0294] Another aspect of the present invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising contacting the single-stranded oligonucleotide as described above with the cells of the subject by 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 cells of the subject, or administering them to the subject.

[0295] Another aspect of the present invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising contacting the oligonucleotide construct as described above, which comprises two single-stranded oligonucleotides by formula (I), (II) (or (IIa)) or (III) (or (IIIa)), with the cells of the subject 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.

[0296] All of the above-described embodiments relating to single-stranded oligonucleotides, nucleotide sequence(s), all variables defined in formula (I) and formula (I), all variables defined in formula (II)-(IIa) and formula (II)-(IIa), all variables defined in formula (III)-(IIIa) and formula (III)-(IIIa), chemical modifications on the nucleotide sequence, the linking group L within the oligonucleotide, the tethering group that covalently links two single-stranded oligonucleotides, and the ligands and ligand conjugations disclosed in the above-described aspects of the present invention related to single-stranded oligonucleotides are suitable for this aspect of the present invention relating to a method for inhibiting the expression of one or more target genes in a subject.

[0297] In some embodiments, the cell is within a subject. In one embodiment, the subject is human. In one embodiment, the subject is a non-human mammal, such as a rhesus monkey, cynomolgous monkey, mouse, or rat.

[0298] In all of the above-described aspects of the present invention, the single-stranded oligonucleotide can inhibit the activity or expression of one or more target genes in the tissue of the subject by at least 15% each, as compared to an appropriate control (e.g., compared to an untreated or placebo-treated subject, or compared to a reference value, such as the target mRNA or protein level in the treated subject measured prior to treatment with the single-stranded oligonucleotide or double-stranded nucleic acid agent). As compared to an appropriate control, it may be able to inhibit the activity or expression of one or more target genes in the tissue of the 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%, at least 95% each. In one embodiment, the appropriate control is an untreated subject. In one embodiment, the appropriate control is a reference value, such as a value obtained in the subject prior to administration of the single-stranded oligonucleotide to the subject.

Brief Description of the Drawings

[0299]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

[0300] The inventors have designed a novel strategy for preparing single-stranded loop oligonucleotides using two chemically modified oligonucleotides that can form intrastrand duplex regions and are connected by a cleavable linker to generate a single-stranded construct. The single-stranded loop oligonucleotides are designed such that the single-stranded construct provides cleavage at a rate suitable for the in vivo effective cleavage into a double-stranded RNAi agent. The single-stranded loop oligonucleotides are synthesized as single strands, self-anneal by sequence complementarity, and are purified as single strands. Delivery ligands such as tris-antennary 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 manufacture and purification of RNAi agents by increasing throughput and decreasing the overall synthesis time, while at the same time providing an improved design such that the effectiveness of the RNAi agent is conserved or improved when cleaved in vivo. Single-Stranded Oligonucleotide Structure Design

[0301] One aspect of the invention is of formula (I): (5'-Z 1 -3')-Q 1 -L-Q 2 -(5'-Z 2 -3') (I) [Wherein: Z 1 is the first oligonucleotide comprising 15 to 100 appropriately modified nucleotides that are substantially complementary to the target gene; Z 2 is the second oligonucleotide comprising 15 to 100 appropriately modified nucleotides 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 which can inhibit the expression of the target gene, wherein at least one nucleotide of formula (I) is a modified nucleotide, relates to a single-stranded oligonucleotide.

[0302] The single-stranded oligonucleotide is formed by connecting two oligonucleotides with a linking group L. Some exemplary single-stranded oligonucleotide constructs are illustrated in Schemes 1 and 2.

[0303]

Chemical formula

[0304]

Chemical formula

[0305] As shown in Schemes 1 and 2, in some embodiments, Z 1 represents the first oligonucleotide that is substantially complementary to the target gene (e.g., the antisense strand); Z 2 is Z 1A second oligonucleotide that is substantially complementary to (e.g., the sense strand). In some embodiments, Z 1 and Z 2 can form an intrastrand double-stranded region between the corresponding nucleotides of Z 1 and Z 2 , and the single-stranded oligonucleotide contains a loop region formed by the linking group L (and possibly Q 1 and Q 2 ). In some embodiments, Q 1 and Q 2 may each independently be absent. In some embodiments, Q 1 and Q 2 may each independently be present as overhangs on the first oligonucleotide Z 1 and the second oligonucleotide Z 2 , respectively. As shown in Schemes 1 and 2, L is a linking group that can contain modified or unmodified nucleotides. In some embodiments, as shown in Scheme 2, L can contain a non-nucleotide-based linker such as Q304. In some embodiments, as shown in Schemes 1 and 2, the nucleotides of the entire single-stranded oligonucleotide (including Z 1 , Z 2 , Q 1 , Q 2 , and L) can 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 further comprises a ligand conjugated by a trivalent branched linker

[0306]

Chemical Formula

[0307] The first oligonucleotide Z 1 and the second oligonucleotide Z 2 each can be at least 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. The first oligonucleotide Z 1 and the second oligonucleotide Z 2 each can have a length of about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, about 10 to about 20 nucleotides, about 15 to about 50 nucleotides, about 15 to about 40 nucleotides, about 15 to about 35 nucleotides, about 15 to about 30 nucleotides, about 15 to about 25 nucleotides, about 15 to about 20 nucleotides, or about 18 to about 20 nucleotides. In one embodiment, the first oligonucleotide Z 1 and the second oligonucleotide Z 2 each are at least 15 nucleotides in length. In one embodiment, the first oligonucleotide Z 1 and the second oligonucleotide Z 2 each are at least 18 nucleotides in length.

[0308] Another aspect of the present invention is 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), [wherein: Z 11 is a first oligonucleotide comprising 15 to 100 appropriately modified nucleotides that are substantially complementary to the target gene; Z 12 is, 11 a second oligonucleotide comprising 10 to 100 appropriately modified nucleotides that are substantially complementary to Z Z 11 and Z 12 can form an intrastrand duplex region containing 7 or more consecutive base pairs; Q S represents 0 to 12 appropriately modified nucleotides, L is any linking group] is a single-stranded oligonucleotide by at least one nucleotide of formula (II) is a modified nucleotide; and at least one nucleotide of formula (III) is a modified nucleotide, where at least one nucleotide at the 3' end of Z 11 in formula (II), and at least one nucleotide at the 5' end of Z 11 in formula (III) are both L and Q S together with Z 11 and Z 12 form a loop region connecting them, relates to a single-stranded oligonucleotide.

[0309] The single-stranded oligonucleotide is formed by connecting two oligonucleotides with an appropriate linking group L.

[0310] In some embodiments, the single-stranded oligonucleotide has the formula (IIa) or formula (IIIa):

[0311]

Chemical formula

[0312]

Chemical formula

[0313]

Chemical formula

[0314]

Chemical formula

[0315]

Chemical formula

[0316] Some exemplary single-stranded oligonucleotides are illustrated in Schemes 1B.1 to 1B.5 and Schemes 2B.1 to 2B.4.

[0317] Some exemplary single-stranded oligonucleotides are illustrated by Schemes 1B.1 to 1B.5, and are defined by Formula (II) or (IIa) as Z 11 and Z 12 ​It may have a direction (e.g., 5'-3' direction) and connections. The PS nucleotide linkages illustrated in each of Schemes 1B.1 to 1B.5 are exemplary and may or may not be present. In certain embodiments, the PS nucleotide linkages illustrated at the 3' and 5' ends are present, but no internal PS nucleotide linkages are present.

[0318]

Chemical Formula

[0319] As shown in Scheme 1B.1, in some embodiments, Z 11 represents a first oligonucleotide that is substantially complementary to a target gene (e.g., the antisense strand); Z 12 is a second oligonucleotide that is substantially complementary to Z 11 (e.g., the sense strand). In some embodiments, Z 11 and Z 12 can form an intrastrand double-stranded region between their corresponding nucleotides, and the single-stranded oligonucleotide contains a loop region LP (possibly including a linker L; not marked). 11 and Z 12 In some embodiments, Q

[0320] may not be present. In some embodiments, Q S is represented by a and b, which may be a spacer and can be any appropriately modified nucleotide that forms a matching or mismatching base pair with those opposite nucleotides of Z S (e.g., the two corresponding nucleotides at positions 17 and 18 in Scheme 1B.1). In one embodiment, both a and b form matching base pairs with those opposite nucleotides of Z 11 . In one embodiment, both a and b form mismatching base pairs with those opposite nucleotides of Z 11 . In one embodiment, one of a and b is Z 11 's opposite nucleotide and the other is a mismatching base pair. In one embodiment, one of a and b is Z11 forms a Watson-Crick base pair with the opposite nucleotide thereof, and the other a and b are Z 11 forms a mismatched base pair with the opposite nucleotide thereof. In one embodiment, b is Z 11 forms a mismatched base pair with the opposite nucleotide thereof (e.g., b is mismatched with the nucleotide at position 17 as shown in Scheme 1B.1). In one embodiment, a is Z 11 forms a mismatched base pair with the opposite nucleotide thereof (e.g., a is mismatched with the nucleotide at position 18 as shown in Scheme 1B.1).

[0321] In some embodiments, the single-stranded oligonucleotide is Z 11 or Z 12 contains one or two phosphorothioate internucleotide link modifications (e.g., internucleotide link modifications between two consecutive phosphorothioate nucleotides) within the first 6 nucleotides or the last 6 nucleotides (i.e., one or two phosphorothioate internucleotide link modifications between nucleotides at the terminal 6 positions from either the 5'-end or the 3'-end of either Z 11 or Z 12 ). In one embodiment, the single-stranded oligonucleotide is Z 11 or Z 12 contains two consecutive phosphorothioate internucleotide link modifications within the first 3 nucleotides or the last 3 nucleotides (i.e., the internucleotide link between the terminal 3 positions from either the 5'-end or the 3'-end of either Z 11 or Z 12 is modified by two consecutive phosphorothioate internucleotide link modifications, e.g., the phosphorothioate internucleotide link modification indicated as a "star" in iii) of Scheme 1B.1).

[0322] In some embodiments, the single-stranded oligonucleotide is Z 11contains one or two phosphorothioate internucleotide linkages (e.g., internucleotide linkages between two consecutive phosphorothioate nucleotides) within the last 8 nucleotides. In one embodiment, the single-stranded oligonucleotide has a length of 23 nucleotides of Z 11 and contains two phosphorothioate internucleotide linkages between nucleotides at positions 16 to 23 (e.g., as shown in Scheme 1B.1, two phosphorothioate internucleotide linkages between nucleotides at positions 16 to 17, 17 to 18, 19 to 20, 20 to 21, and 21 to 22 of Z 11 ).

[0323] In some embodiments, the single-stranded oligonucleotide contains one or two phosphorothioate internucleotide linkages (e.g., internucleotide linkages between two consecutive phosphorothioate nucleotides) within the first 3 nucleotides of Z 12 (e.g., as shown in Scheme 1B.1, one or two phosphorothioate internucleotide linkages between nucleotides at positions 1 to 2 and / or 2 to 3 of Z 12 ). In one embodiment, the single-stranded oligonucleotide contains two consecutive phosphorothioate internucleotide linkages between nucleotides at positions 1 to 2 and 2 to 3 of Z 12 , two consecutive phosphorothioate internucleotide linkages between the last 3 nucleotides (e.g., positions 14 to 15 and 15 to 16) of Z 12 , and two consecutive phosphorothioate internucleotide linkages between nucleotides at positions 1 to 2 and 2 to 3 of Z 11 as shown in ii) of Scheme 1B.1.

[0324] In some embodiments, as shown in ii) of Scheme 1B.1, the single-stranded oligonucleotide has, at the 5'-end of the nucleotide sequence (e.g., Z 11 and / or Z 12 ), a 5'-phosphate modification or a 5'-phosphate mimetic modification as described herein (e.g., as shown in ii) of Scheme 1B.1, Z 11may include 5'-terminal vinyl phosphonate (5'-VP) at the 5'-end.

[0325] In some embodiments, the single-stranded oligonucleotide may further include one or more ligands (e.g., lipophilic moieties for extrahepatic delivery as described herein). In some embodiments, one or more lipophilic moieties are independently conjugated to one or more internal positions (i.e., non-terminal positions) of Z 11 . In one embodiment, one or more lipophilic moieties are independently conjugated to one or more of the 11th, 12th, and 13th positions from the 5'-end of Z as shown in ii) of Scheme 1B.1. In one embodiment, one or more lipophilic moieties are independently conjugated to one or more internal positions of Z excluding the 2nd or 14th position. In one embodiment, one or more lipophilic moieties are, as shown in ii) of Scheme 1B.1, Q 11 paired with the 11th, 12th, and 13th positions from the 5'-end of Z 11 and / or are independently conjugated to one or more positions of Z 11 excluding the positions of S and / or Z 12 , and one or more positions of Z 12 and / or Q S .

[0326] In some embodiments, the single-stranded oligonucleotide may further include 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) are conjugated to the 3'-end of Z 11 or Z 12 as shown in iii) of Scheme 1B.1, or to an internal position of Z 11 or Z 12 . In some embodiments, one or more targeting ligands (e.g., carbohydrate-based ligands) are conjugated to one or more internal positions of Z excluding the 2nd or 14th position. 11is 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 Scheme 1B.1 iii) 12 to the 3’ end of

[0327] In some embodiments, if a single-stranded oligonucleotide contains terminal conjugation of a ligand to a 5’ or 3’ terminal nucleotide, or terminal conjugation of a depurinated nucleotide, inverted nucleotide, or inverted depurinated nucleotide to a 5’ or 3’ terminal nucleotide, then at that terminus, the above internucleotide linkage modification to the terminal nucleotide can be removed (e.g., by conjugation of a ligand to the 3’ end of Z as shown in Scheme 1B.1 iii), the modification of the internucleotide linkage between the terminal 6th or 3rd nucleotide to the 3’ end of Z 12 can be removed). 12 by conjugation of a ligand to the 3’ end of Z

[0328]

Chemical formula

[0329] In some embodiments, as shown in Scheme 1B.2, Z 11 contains 19 - 23 appropriately modified nucleotides, Z 12 contains 12 - 16 appropriately modified nucleotides, and Q S contains 2 appropriately modified nucleotides.

[0330] In some embodiments, as shown in Scheme 1B.2, the double-stranded region formed by Z 11 and Z 11 and Z 12 at a non-loop terminus (e.g., the 5’ end of Z) has blunt ends.

[0331]

Chemical formula

[0332] In some embodiments, as shown in Scheme 1B.3, Z 11 comprises from 19 to 23 optionally modified nucleotides, and Z 12 comprises from 12 to 16 optionally modified nucleotides, and Q S comprises 2 optionally modified nucleotides.

[0333] In some embodiments, as shown in Scheme 1B.3, the 3 to 5 terminal nucleotides of Z S connected to L, Q 12 , or Z 11 contain a modification 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, the 5 terminal nucleotides of Z S connected to L, Q 12 , or Z 11 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 [wherein: # is the bond to Z 11 , ** is the bond to L, Q S , or Z 12 ​ dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents a ribonucleotide, and mN represents a 2'-O-methylnucleotide] has a modification selected from the group consisting of.

[0334] In some embodiments, as shown in Scheme 1B.3, L, Q S , or Z 12 The three terminal nucleotides of Z connected to 11 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- ** has a modification independently selected from the group consisting of.

[0335] In one embodiment, as shown in Scheme 1B.3, the single-stranded oligonucleotide has two consecutive phosphorothioate internucleotide linkage modifications between the nucleotides at positions 1-2 and 2-3 of Z 11 , and two consecutive phosphorothioate internucleotide linkage modifications between the last three nucleotides of Z 12 .

[0336] In one embodiment, as shown in Scheme 1B.3, the single-stranded oligonucleotide has 6 terminal phosphorothioate internucleotide linkages; Z 11 Two consecutive phosphorothioate internucleotide linkages between nucleotides at positions 1-2 and 2-3 of Z 12 Two consecutive phosphorothioate internucleotide linkages between the last 3 nucleotides of Z 12 And two consecutive phosphorothioate internucleotide linkages between nucleotides at positions 1-2 and 2-3 of Z

[0337] In some embodiments, as shown in Scheme 1B.3, the second oligonucleotide Z 12 Optionally contains at least one motif of three consecutive 2'-F modifications together with Q S And the nucleotide following the motif is not 2'-F modified. In some embodiments, the positions of the motif of three consecutive modifications are as follows: The motif is in Q S At positions 1 and 2 of Z 12 And Z 11 May be 19 nucleotides in length; The motif is in Z 12 At positions 1, 2 and 3 of Z 11 And Z The motif is in Z 12 At positions 2, 3 and 4 of Z 11 And Z The motif is in Z 12 At positions 3, 4 and 5 of Z 11 And Z The motif is in Z 12 At positions 4, 5 and 6 of Z 11 And Z Is characterized by one of

[0338] In some embodiments, as shown in Scheme 1B.3, the first oligonucleotide Z 11contains modifications that are not 2'-O-methyl at the 2nd and 14th positions. In one embodiment, as shown in Scheme 1B.3, the first oligonucleotide Z 11 contains a 2'-F modification at the 14th position.

[0339] In some embodiments, as shown in Scheme 1B.3, the first oligonucleotide Z 11 contains one or more 2'-deoxy (DNA) modifications at positions 2, 5, 7, and 12.

[0340] In some embodiments, as shown in Scheme 1B.3, Z 11 and Z 12 all remaining modifications are 2'-O-methyl modifications.

[0341]

Chemical formula

[0342] In some embodiments, as shown in Scheme 1B.4, Z 11 contains appropriately modified nucleotides from 19 to 23, Z 12 contains appropriately modified nucleotides from 12 to 16, and Q S contains appropriately modified nucleotides at 2.

[0343] In some embodiments, as shown in Scheme 1B.4, L, Q S or Z 12 connected to Z 11 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 to Z 11 5 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 [wherein: # is a bond to Z 11 and ** is a bond to L, Q S or Z 12 and dN represents a 2'-deoxynucleotide, fN represents a 2'-deoxy-2'-fluoronucleotide, rN represents a ribonucleotide, mN represents a 2'-O-methyl nucleotide] having a modification selected from the group consisting of.

[0344] In some embodiments, as shown in Scheme 1B.4, the three terminal nucleotides of Z connected to L, Q S or Z 12 are 2'-fluoro, 2'-deoxy, and 2'-OH, for example 11 #-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- ** has a modification independently selected from the group consisting of.

[0345] In one embodiment, as shown in Scheme 1B.4, the single-stranded oligonucleotide has two consecutive phosphorothioate internucleotide linkages between the nucleotides at positions 1 to 2 and 2 to 3 of Z 11 , and two consecutive phosphorothioate internucleotide linkages between the last three nucleotides of Z 12 .

[0346] In one embodiment, as shown in Scheme 1B.4, the single-stranded oligonucleotide has six terminal phosphorothioate internucleotide linkages; two consecutive phosphorothioate internucleotide linkages between the nucleotides at positions 1 to 2 and 2 to 3 of Z 11 , two consecutive phosphorothioate internucleotide linkages between the last three nucleotides of Z 12 , and two consecutive phosphorothioate internucleotide linkages between the nucleotides at positions 1 to 2 and 2 to 3 of Z 12 .

[0347] In some embodiments, as shown in Scheme 1B.4, the second oligonucleotide Z 12 optionally contains at least one motif of three consecutive 2'-F modifications, together with Q S , and the nucleotide following the motif is not 2'-F modified. In some embodiments, the positions of the motif of three consecutive modifications are as follows: The motif is at positions 1 and 2 of Q S , Z 12 , and Z 11 may be 19 nucleotides in length; The motif is at positions 1, 2, and 3 of Z 12 , Z11 may be 20 nucleotides in length; The motif is at positions 2, 3, and 4 of Z 12 and Z 11 may be 21 nucleotides in length; The motif is at positions 3, 4, and 5 of Z 12 and Z 11 may be 22 nucleotides in length; or The motif is at positions 4, 5, and 6 of Z 12 and Z 11 may be 23 nucleotides in length and is characterized by one of .

[0348] In some embodiments, as shown in Scheme 1B.4, Z 12 optionally contains, together with Q S a 2'-O-methyl or 2'-F modification at a position that is two positions before the 2-position of a motif of three consecutive 2'-F modifications, provided that the position is not part of Z 11 (when the motif starts at position n, position n - 2). In one embodiment, as shown in Scheme 1B.4, Z 12 optionally contains, together with Q S a 2'-F modification at a position that is two positions before the 2-position of a motif of three consecutive 2'-F modifications, provided that the position is not part of Z 11 (when the motif starts at position n, position n - 2).

[0349] In some embodiments, as shown in Scheme 1B.4, the first oligonucleotide Z 11 contains a modification other than 2'-O-methyl at positions 2 and 14. In one embodiment, as shown in Scheme 1B.4, the first oligonucleotide Z 11 contains a 2'-F modification at position 14.

[0350] In some embodiments, as shown in Scheme 1B.4, the first oligonucleotide Z 11 contains one or more 2'-F modifications at positions 2, 6, 8, 9, 14, and 16.

[0351] In some embodiments, as shown in Scheme 1B.4, Z 11 and all remaining modifications of Z 12 are 2'-O-methyl modifications.

[0352]

Chemical formula

[0353] 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 a bond to Z 11 , ** is a bond to Q S or Z 12 ; n is 3 to 12; each N is, independently, a linking 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.

[0354] In some embodiments, as shown in Scheme 1B.5, all linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 . In some embodiments, as shown in Scheme 1B.5, one or more linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 , and one or more linking monomers of L (e.g., Q304) are not in the loop region. In some embodiments, as shown in Scheme 1B.5, one or more linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 , and one or more linking monomers of L (e.g., Q304) are not in the loop and are Q Sis connected to (a). In some embodiments, as shown in Scheme 1B.5, one or more linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 , and one or more linking monomers of L (e.g., Q304) are not in the loop but are connected to Z 12 .

[0355] In some embodiments, one or more linking moieties (N) in L can be appropriately modified nucleotides. In some embodiments, one or more linking moieties (N) in L are 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).

[0356] In some embodiments, one or more linking moieties (N) in L are 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.

[0357] In some embodiments, as shown in Scheme 1B.5, L contains a triplet of Q304.

[0358] In some embodiments, as shown in Scheme 1B.4, the single-stranded oligonucleotide has two consecutive phosphorothioate internucleotide linkages between the nucleotides at positions 1-2 and 2-3 of Z 11 , two consecutive phosphorothioate internucleotide linkages between the last three nucleotides of Z 12 , and Z 12Contains two consecutive phosphorothioate internucleotide link modifications between the nucleotides at positions 1 to 2 and 2 to 3.

[0359] In some embodiments, as shown in Scheme 1B.5, the single-stranded oligonucleotide contains one or two phosphorothioate internucleotide link modifications (e.g., two consecutive phosphorothioate internucleotide link modifications) within the last 5, 6, or 7 nucleotides of Z 11 .

[0360] In one embodiment, as shown in Scheme 1B.5, the single-stranded oligonucleotide contains six terminal phosphorothioate internucleotide link modifications; two consecutive phosphorothioate internucleotide link modifications between the nucleotides at positions 1 to 2 and 2 to 3 of Z 11 ; two consecutive phosphorothioate internucleotide link modifications between the last 3 nucleotides of Z 12 ; and two consecutive phosphorothioate internucleotide link modifications between the nucleotides at positions 1 to 2 and 2 to 3 of Z 12 .

[0361] In one embodiment, as shown in Scheme 1B.5, the single-stranded oligonucleotide contains eight terminal phosphorothioate internucleotide link modifications; two consecutive phosphorothioate internucleotide link modifications between the nucleotides at positions 1 to 2 and 2 to 3 of Z 11 ; two consecutive phosphorothioate internucleotide link modifications between the last 3 nucleotides of Z 12 ; two consecutive phosphorothioate internucleotide link modifications between the nucleotides at positions 1 to 2 and 2 to 3 of Z 12 ; and two consecutive phosphorothioate internucleotide link modifications within the last 5, 6, or 7 nucleotides of Z 11 .

[0362] Some exemplary single-stranded oligonucleotides are of formula (III) or (IIIa) as illustrated by Schemes 2B.1 to 2B.4, of Z 11and Z 12 may have a direction (e.g., 5'-3' direction) and a connection.

[0363] [Chemical formula]

[0364] [Chemical formula]

[0365] [Chemical formula]

[0366] [Chemical formula]

[0367] In some embodiments, Z 11 comprises appropriately modified nucleotides from 19 to 23, and Z 12 comprises appropriately modified nucleotides from 16 to 19, and Q S comprises two appropriately modified nucleotides, and may or may not be present.

[0368] In some embodiments, as shown in Scheme 2B.1, Z 11 comprises an appropriately modified nucleotide of 23, and Z 12 comprises appropriately modified nucleotides from 16 to 19. In some embodiments, as shown in Scheme 2B.2, Z 11 comprises an appropriately modified nucleotide of 21, and Z 12 comprises appropriately modified nucleotides from 14 to 17. In some embodiments, as shown in Scheme 2B.3, Z 11 comprises an appropriately modified nucleotide of 23, and Z 12 comprises appropriately modified nucleotides from 18 to 21. In some embodiments, as shown in Scheme 2B.4, Z 11comprises 21 appropriately modified nucleotides, and Z 12 comprises 16 - 19 appropriately modified nucleotides.

[0369] In some embodiments, as shown in Schemes 2B.3 and 2B.4, the double - stranded region formed by Z 11 at the non - loop end (e.g., the 3’ end of Z 11 and Z 12 has blunt ends.

[0370] In some embodiments, Z 11 at the non - loop end has an overhang of 1 - 3 nucleotides in length. In one embodiment, Z 11 at the non - loop end has an overhang of 2 nucleotides in length (e.g., the 3’ end of Z 11 as shown in Schemes 2B.1 and 2B.2). In one embodiment, as shown in Schemes 2B.1 and 2B.2, Z 11 at the non - loop end has an overhang of 2 nucleotides in length and has a phosphorothioate internucleotide linkage between the two overhang nucleotides.

[0371] In one embodiment, as shown in Schemes 2B.1 and 2B.2, Z 11 at the non - loop end has an overhang of 2 nucleotides in length (e.g., the 3’ end of Z 11 ), has two phosphorothioate internucleotide linkages between the terminal 3 nucleotides (e.g., the 3’ end of Z 11 ), two of the 3 nucleotides are overhang nucleotides, and the third is the paired nucleotide next to the overhang nucleotide.

[0372] In some embodiments, as shown in Schemes 2B.1 and 2B.2, the single - stranded oligonucleotide is within the first 4 nucleotides of Z 11 or within Z 12contains one or two phosphorothioate internucleotide link modifications (e.g., a modification of the linkage between two consecutive phosphorothioate nucleotides) within the first three nucleotides of

[0373] In some embodiments, as shown in Schemes 2B.1 - 2B.4, the single-stranded oligonucleotide contains six terminal phosphorothioate internucleotide link modifications; Z 12 two consecutive phosphorothioate internucleotide link modifications between nucleotides at positions 1 - 2 and 2 - 3 of 11 two consecutive phosphorothioate internucleotide link modifications between nucleotides within the first four nucleotides of 11 and two consecutive phosphorothioate internucleotide link modifications between nucleotides within the last three nucleotides of

[0374] In some embodiments, as shown in Schemes 2B.1 - 2B.4, the single-stranded oligonucleotide contains eight terminal phosphorothioate internucleotide link modifications; Z 12 two consecutive phosphorothioate internucleotide link modifications between nucleotides at positions 1 - 2 and 2 - 3 of 12 two consecutive phosphorothioate internucleotide link modifications between nucleotides within the last three nucleotides of 11 two consecutive phosphorothioate internucleotide link modifications between nucleotides within the first four nucleotides of 11 and two consecutive phosphorothioate internucleotide link modifications between nucleotides within the last three nucleotides of

[0375] In some embodiments, if the single-stranded oligonucleotide contains a terminal conjugation of a ligand to a 5' or 3' terminal nucleotide, or a terminal conjugation of an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide to a 5' or 3' terminal nucleotide, then at that terminus, the above-described internucleotide link modification to the terminal nucleotide can be removed (e.g., as shown in Schemes 2B.1 - 2B.4, Z 12Conjugation of the ligand to the 5' end of Z 12 can remove one or two phosphorothioate internucleotide linkages between nucleotides at positions 6 or 3 at the 5' end of

[0376] In some embodiments, as shown in Schemes 2B.1 - 2B.4, the intrastrand duplex region formed by Z 11 and Z 12 can contain all consecutive base pairs or can contain a maximum of 3 (e.g., 0, 1, 2, or 3) mismatched base pairs. In one embodiment, Z 12 can contain one nucleotide that forms a mismatched base pair with the opposite nucleotide of Z 11 (e.g., the last nucleotide of Z 12 , or the (n - 1)th nucleotide if the last nucleotide is the nth nucleotide).

[0377] In some embodiments, as shown in Schemes 2B.1 - 2B.4, the 3 - 5 terminal nucleotides of L, Q S , or Z 12 connected to Z 11 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, the 5 terminal nucleotides of L, Q S , or Z 12 connected to Z 11 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.

[0378] In some embodiments, as shown in Schemes 2B.1 - 2B.4, L, Q S , or Z 12 connected to Z 11 has three terminal nucleotides of 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- ** has a modification independently selected from the group consisting of.

[0379] In some embodiments, L is present in Formula (II) (or IIa) or Formula III (or IIIa) and contains a linking moiety represented by the formula: #-(N) n - ** In this formula, # is a bond to Z 11 , ** is Q S or Z 12a linkage to; n is from 3 to 12; each N is, independently, a linking monomer having a chain length of 3 or more atoms. 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 3.

[0380] In some embodiments, as shown in Schemes 2B.1 - 2B.4, all of the linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 . In some embodiments, as shown in Schemes 2B.1 - 2B.4, one or more linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 , and one or more linking monomers of L (e.g., Q304) are not in the loop region. In some embodiments, as shown in Schemes 2B.1 - 2B.4, one or more linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 , and one or more linking monomers of L (e.g., Q304) are not loops but are connected to Q S (a). In some embodiments, as shown in Schemes 2B.1 - 2B.4, one or more linking monomers of L (e.g., Q304), together with LP, form a loop between W(Z 11 ) and Z 12 , and one or more linking monomers of L (e.g., Q304) are not loops but are connected to Z 12 .

[0381] In some embodiments, one or more linking moieties (N) in L can be appropriately modified nucleotides. In some embodiments, one or more linking moieties (N) in L can independently be selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methyl nucleotide (mN), and 2'-ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide).

[0382] In some embodiments, one or more linking moieties (N) in L can independently be 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.

[0383] In one embodiment, as shown in Schemes 2B.1 - 2B.4, L contains a triplet of Q304.

[0384] The single-stranded oligonucleotide sequence can be a substrate cleavable by DICER.

[0385] 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 linked. In some embodiments, the two single-stranded oligonucleotides are covalently linked via a tethering group. Exemplary tethering groups and exemplary processes for covalently linking two single-stranded oligonucleotides to form an oligonucleotide construct are shown in Schemes 7.1 - 7.4 below.

[0386] Certain embodiments of the present invention relate to a linker design for connecting two oligonucleotides to form a single-stranded oligonucleotide. Certain embodiments of the present invention relate to a tethering group design for connecting two oligonucleotides to form an oligonucleotide construct (i.e., a bivalent chromosome style). Linker / Tether

[0387] The linker / tether is contained in the linker group L of the single-stranded oligonucleotide and can connect two oligonucleotides to form a single-stranded oligonucleotide.

[0388] The linker / tether is contained in the tethering group of the oligonucleotide construct (i.e., bivalent chromosome style) and can connect two single-stranded oligonucleotides to form an oligonucleotide construct.

[0389] The linker / tether can also be used, for example, to connect a ligand to a single-stranded oligonucleotide via a carrier.

[0390] The terms "linker", "linkage", "linker group", "linking moiety", and "tether" can be used interchangeably.

[0391] The linker group L may contain a plurality of linkers / tethers, which may be the same or different from each other.

[0392] The linker group L of the single-stranded oligonucleotide can be a nucleotide-based or non-nucleotide-based linker. The linker group L can be a stable linker that is stable in a body fluid (e.g., in plasma or artificial cerebrospinal fluid). Alternatively, the linker group L can be a cleavable linker (e.g., a bio-cleavable linker).

[0393] The linker / tether can be connected to a ligand at a "tethering junction point (TAP)". The linker / tether can be any C 1 ~C 100 carbon-containing moiety (e.g., C1 ~C 75 、C 1 ~C 50 、C 1 ~C 20 、C 1 ~C 10 ;C 1 、C 2 、C 3 、C 4 、C 5 、C 6 、C 7 、C 8 、C 9 、 or C 10 ) and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of the terminal amino or amide (NHC(O)-) group of the linker / tether and can be used as a point of attachment for the ligand. Non-limiting examples of the linker / tether (underlined) are TAP- (CH 2 ) n NH- ;TAP- C(O)(CH 2 ) n NH- ;TAP- NR’’’’(CH 2 ) n NH- 、TAP- C(O)-(CH 2 ) n -C(O)- ;TAP- C(O)-(CH 2 ) n -C(O)O- ;TAP- C(O)-O- ;TAP- C(O)-(CH 2 ) n -NH-C(O)- ;TAP- C(O)-(CH 2 ) n - ;TAP- C(O)-NH- ;TAP- C(O)- ;TAP- (CH 2 ) n -C(O)-;TAP- (CH 2 ) n -C(O)O- ;TAP- (CH 2 ) n - ; or TAP- (CH 2 ) n -NH-C(O)- including, where n is from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R'''' is C 1 ~C 6 alkyl. Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen can form part of a terminal oxyamino group, e.g., -ONH 2 , or a hydrazino group, -NHNH 2 . The linker / tether can be substituted, for example, by hydroxy, alkoxy, perhaloalkyl, and / or one or more additional heteroatoms, e.g., N, O, or S, can be inserted. Preferred tethered ligands are, for example, TAP- (CH 2 ) n NH(LIGAND) ;TAP- C(O)(CH 2 ) n NH(LIGAND) ;TAP- NR’’’’(CH 2 ) n NH(LIGAND) ;TAP- (CH 2 ) n ONH(LIGAND) ;TAP- C(O)(CH 2 ) n ONH(LIGAND) ;TAP- NR’’’’(CH 2 ) n ONH(LIGAND) ;TAP- (CH2 ) n NHNH 2 (LIGAND) , TAP- C(O)(CH 2 ) n NHNH 2 (LIGAND) ; TAP- NR’’’’(CH 2 ) n NHNH 2 (LIGAND) ; TAP- C(O)-(CH 2 ) n -C(O)(LIGAND) ; TAP- C(O)-(CH 2 ) n -C(O)O(LIGAND) ; TAP- C(O)-O(LIGAND) ; TAP- C(O)-(CH 2 ) n -NH-C(O)(LIGAND) ; TAP- C(O)-(CH 2 ) n (LIGAND) ; TAP- C(O)-NH(LIGAND) ; TAP- C(O)(LIGAND) ; TAP- (CH 2 ) n -C(O)(LIGAND) ; TAP- (CH 2 ) n -C(O)O(LIGAND) ; TAP- (CH 2 ) n (LIGAND) ; or TAP- (CH 2 ) n -NH-C(O)(LIGAND) may include. In some embodiments, the amino-terminal linker / tether (e.g., NH 2 , ONH 2 , NH 2 NH 2) can form an imino bond (i.e., C=N) with a ligand. In some embodiments, the amino-terminal linker / tether (e.g., NH 2 , ONH 2 , NH 2 NH 2 ) can be acylated, for example, by C(O)CF 3 .

[0394] In some embodiments, the linker / tether can be terminated by a mercapto group (i.e., SH) or an olefin (e.g., CH=CH 2 ). For example, the tether can be TAP- (CH 2 ) n -SH , TAP- C(O)(CH 2 ) n SH , TAP- (CH 2 ) n -(CH=CH 2 ) , or TAP- C(O)(CH 2 ) n (CH=CH 2 ) , where n can be as described elsewhere in this specification. The tether can be optionally substituted, for example, by hydroxy, alkoxy, perhaloalkyl, and / or one or more additional heteroatoms, such as N, O, or S, can be optionally inserted. The double bond can be cis or trans, or E or Z.

[0395] 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, aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, such as an NHS ester, or a pentafluorophenyl ester. Preferred linkers / tethers (underlined) are TAP- (CH 2 ) n CHO ; TAP- C(O)(CH 2 ) n CHO ; or TAP- NR’’’’(CH 2 ) n CHO (where n is from 1 to 6 and R'''' is C 1 ~C 6 alkyl); or TAP- (CH 2 ) n C(O)ONHS ; TAP- C(O)(CH 2 ) n C(O)ONHS ; or TAP- NR’’’’(CH 2 ) n C(O)ONHS (where n is from 1 to 6 and R'''' is C 1 ~C 6 alkyl); TAP- (CH 2 ) n C(O)OC 6 F 5 ; TAP- C(O)(CH 2 ) n C(O)OC 6 F 5 ; or TAP- NR’’’’(CH 2 )n C(O)OC 6 F 5 (where n is from 1 to 11, and R'''' is C 1 ~C 6 alkyl); or -(CH 2 ) n CH 2 LG ; TAP- C(O)(CH 2 ) n CH 2 LG ; or TAP- NR’’’’(CH 2 ) n CH 2 LG [where n is described elsewhere in this specification, and R'''' is C 1 ~C 6 alkyl (LG is a group that can release a group such as a halide, mesylate, tosylate, nosylate, brosylate)]. Tethering can be performed by coupling a nucleophilic group of the ligand, such as a thiol or amino group, with an electrophilic group of the tether.

[0396] In other embodiments, it may be desirable for the monomer to include a phthalimide group (K) at the terminal position of the linker / tether.

[0397]

Chemical formula

[0398] In other embodiments, other protected amino groups, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (for example, the aryl moiety can be ortho-nitrophenyl or ortho, para-dinitrophenyl), can be at the terminal position of the linker / tether.

[0399] Any linker / tether described herein may further include one or more additional linking groups such as -O-(CH 2 ) n -, -(CH 2 ) n -SS-, -(CH 2 ) n -, or -(CH=CH)-.

[0400] Cleavable linker / tether In some embodiments, at least one linker / tether can be a redox-cleavable linker, an acid-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, a peptidase-cleavable linker, or an endosome-cleavable linker.

[0401] In one embodiment, at least one linker / tether can be a reductively cleavable linker (e.g., a disulfide group).

[0402] In one embodiment, at least one linker / tether can be an acid-cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group).

[0403] In one embodiment, at least one linker / tether can be an esterase-cleavable linker (e.g., an ester group).

[0404] In one embodiment, at least one linker / tether can be a phosphatase-cleavable linker (e.g., a phosphate group).

[0405] In one embodiment, at least one linker / tether can be a peptidase-cleavable linker (e.g., a peptide bond).

[0406] In one embodiment, at least one linker / tether can be an endosome-cleavable linker (or protease-cleavable linker, such as 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).

[0407] Cleavable linking groups are sensitive to a cleaving agent, such as the presence of pH, redox ability, or degradable molecules. Generally, the cleaving agent is found at a higher frequency, or at a higher level or activity, inside the cell compared to in serum or blood. Examples of such degrading agents are redox agents selected for a particular substrate, or redox agents without substrate specificity, such as mercaptans that are present intracellularly and can degrade a redox-cleavable linking group by reduction; esterases; agents that can create an endosome, or an acidic environment, such as an acidic environment resulting in a pH of 5 or less; general acids, peptidases (which can be substrate-specific), and redox agents including oxidases or reductases or reducing agents such as enzymes that can hydrolyze or degrade an acid-cleavable linking group by acting as phosphatases.

[0408] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. The pH of human serum is 7.4, but the average intracellular pH is slightly lower, in the range of about 7.1 - 7.3. Endosomes have a strongly acidic pH in the range of 5.5 - 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some tethers are cleaved at a preferred pH, thereby having a linking group that releases the iRNA agent from an intracellular ligand (such as a targeting or cell-permeable ligand, such as cholesterol) or into a desired cellular compartment.

[0409] The chemical conjugation (e.g., linker) that links the ligand to the iRNA agent may include a disulfide bond. When the iRNA agent / ligand complex is taken up by cells via endocytosis, the acidic environment of the endosome causes cleavage of the disulfide bond, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. 19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002). The ligand can be a targeting ligand or a second therapeutic agent that can supplement the therapeutic effect of the iRNA agent.

[0410] The tether can include a linker that is cleaved by a specific enzyme. The type of linker incorporated into the tether can depend on the cell targeted by the iRNA agent. For example, an iRNA agent that targets mRNA in liver cells can be conjugated to a tether containing an ester group. Since liver cells are rich in esterases, the tether will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Cleavage of the tether can 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 cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0411] A tether containing a peptide bond can be conjugated to the iRNA agent and targeted to cell types rich in peptidases, such as liver cells and synovial cells. For example, an iRNA agent that targets synovial cells, such as for the treatment of an inflammatory disease (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond.

[0412] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a cleaving agent (or cleavage conditions) to cleave the candidate linking group. It may also be desirable to examine the ability of a candidate cleavable linking group to resist cleavage in the blood or upon contact with other non-target tissues, such as the tissues to which the iRNA agent is exposed when administered to a subject. It may also be desirable to examine the ability of a candidate cleavable linking group to resist cleavage in the blood or upon contact with other non-target tissues. Thus, the relative sensitivity to cleavage between a first condition selected to indicate cleavage within a target cell and a second condition selected to indicate cleavage within other tissues or in a body fluid, such as in blood or serum, can be determined. The evaluation can be carried out in a cell-free system, cells, cell cultures, organ cultures or tissue cultures, or in whole animals. It can be useful to perform an initial evaluation under cell-free or culture conditions and confirm by further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at a rate that is at least 2, 4, 10 or 100 times faster in cells (or in in vivo conditions selected to mimic intracellular conditions) compared to in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0413] The cleavable linker can be cleaved in various tissues and cell structures, such as homogenates of any type of cell, Tritosomes, cytosol, or endosomes, such as liver homogenate, liver Tritosomes, liver lysosomes, liver cytosol, liver endosomes, brain homogenate, brain Tritosomes, brain lysosomes, brain cytosol, or brain endosomes.

[0414] Redox-cleavable linking group One class of cleavable linking groups are redox-cleavable linking groups that are cleaved upon reduction or oxidation. An example of a reductive-cleavable linking group is a disulfide linking group (-S-S-). The methods described herein can be considered to determine whether a candidate cleavable linking group is a suitable "reductive-cleavable linking group" or, for example, suitable for use with a particular iRNA moiety and a particular targeting agent. For example, a candidate substance can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., within target cells. A candidate substance can also be evaluated under conditions selected to mimic conditions in blood or serum. In a preferred embodiment, a candidate compound is cleaved up to 10% at most in blood. In a preferred embodiment, a useful candidate compound is degraded in cells (or in in vivo conditions selected to mimic intracellular conditions) at a rate that is at least 2, 4, 10 or 100 times faster compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of a candidate compound is determined using a standard enzyme reaction rate assay under conditions selected to mimic an intracellular medium and can be compared to conditions selected to mimic an extracellular medium.

[0415] Phosphate-based cleavable linking group Phosphate-based linking groups are cleaved by agents that decompose or hydrolyze phosphate groups. In cells, examples of agents that cleave phosphate groups are enzymes such as phosphatases within the cell. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidate linkages can be evaluated using methods similar to those described above.

[0416] Acid-cleavable linking group An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by an agent such as an enzyme that can act as a general acid. Inside the cell, specific low-pH organelles such as endosomes and lysosomes provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. The acid-cleavable group may have the general formula, -C=NN-, C(O)O, or -OC(O). A preferred embodiment is an embodiment 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.

[0417] Ester-based linking group Ester-based linking groups are cleaved by enzymes such as intracellular esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkynylene groups. The ester-cleavable linking group has the general formula, C(O)O-, or -OC(O)-. These candidate linking groups can be evaluated using methods similar to those described above.

[0418] Peptide-based cleavage group Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases within cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids so as to result in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavage groups do not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to result in peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to result in peptides and proteins and do not include the entire amide functional group. The peptide-cleavable linking group has the general formula, -NHCHR 1 C(O)NHCHR 2 C(O)- [wherein, R 1 and 2 are the R groups of two adjacent amino acids]. These candidates can be evaluated using a method similar to that described above. Biologically cleavable linker / tether

[0419] The linker can also include a biologically cleavable linker, which is a nucleotide and non-nucleotide linker or a combination thereof that connects two parts of a molecule. For example, a biologically cleavable linker can be used as part of a linking group L that connects two oligonucleotides of a single-stranded oligonucleotide. In some embodiments, a slight electrostatic or stacking interaction between two individual nucleotide sequences can represent the linker.

[0420] Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and their derivatives, aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0421] In some embodiments, at least one linker (tether) is a biodegradable linker selected from the group consisting of DNA, RNA, disulfide, amide, functional monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0422] In some embodiments, a cleavable linker (or biodegradable linker) contains one or more carbohydrate (saccharide) moieties and / or peptide linkers. A cleavable linker (or biodegradable linker) can be used to connect two nucleotide sequences or oligonucleotides, connect a nucleotide sequence or oligonucleotide to a ligand, or connect a ligand to an endosomal cleaving agent.

[0423] In some embodiments, a biodegradable carbohydrate linker has the following characteristics: i) The biodegradable carbohydrate linker can have 1 to 10 saccharide units; ii) The saccharide moiety has at least one anomeric linkage capable of connecting two nucleotide sequences or oligonucleotides; iii) When two or more saccharides are present, these nucleotide sequences or oligonucleotides can be linked via a 1-3, 1-4, or 1-6 saccharide linkage; iv) When two or more saccharides are present, these nucleotide sequences or oligonucleotides can also be linked via an alkyl chain. It has one or more of the above.

[0424] Exemplary biodegradable linkers are

[0425]

Chemical Structure

[0426] In some embodiments, the cleavable linker (or bio-cleavable linker) is the following group:

[0427]

Chemical formula

[0428] In some embodiments, the endosome-cleavable linker comprises two or more of the above saccharide units.

[0429] In some embodiments, the endosome-cleavable linker comprises 1 to 10 saccharide units.

[0430] In some embodiments, the endosome-cleavable linker comprises 2 to 10 saccharide units. In some embodiments, the saccharide units of the endosome-cleavable linker are selected from the group consisting of Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316 and Q317.

[0431] In some embodiments, the endosome-cleavable linker comprises 2, 3, or 4 saccharide units. In some embodiments, the saccharide units are selected from the group consisting of Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316 and Q317. For example, the saccharide unit can be Q304.

[0432] In one embodiment, the endosome-cleavable linker is -Q303Q303-, -Q303Q303Q303-, -Q303Q303Q303Q303, -Q304Q304-, -Q304Q304Q304-, -Q304Q304Q304Q304, -Q305Q305-, -Q305Q305Q305-, -Q306Q306-, -Q306Q306Q306, -Q312Q312-, -Q312Q312Q312-, -Q313Q313-, -Q313Q313Q313, -Q314Q314-, -Q314Q314Q314-, -Q315Q315-, -Q315Q315Q315, -Q316Q316-, -Q316Q316Q316-, -Q317Q317, or -Q317Q317Q317 including.

[0433] In one embodiment, the endosome-cleavable linker is

[0434]

Chemical formula

[0435] In one embodiment, the endosome-cleavable linker is -Q198Q48Q303Q303Q48-, -Q198Q303Q48Q303-, -Q198Q48Q303Q303Q48, -Q198Q303Q48Q303-, -Q198Q303Q303Q303Q303-, -Q198Q303Q303Q303, -Q198Q303Q303-, -Q198Q304Q304Q304Q304-, -Q198Q304Q304Q304, -Q198Q304Q304-, -Q198Q48Q303Q303Q48-, -Q198Q303Q48Q303, -Q198Q303Q303-, -Q48Q303Q303Q48-, or -Q303Q48Q303 including.

[0436] Further discussion of the biodegradable linker can be found in WO2018136620, the entire contents of which are incorporated herein by reference. Carrier

[0437] In certain embodiments, the linking group L that connects two oligonucleotides of a single-stranded oligonucleotide 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 carrier can replace one or more nucleotides (plural available).

[0438] The carrier can be a cyclic group or an 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, pyridazinonyl, tetrahydrofuryl, and decalinyl. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0439] In some embodiments, the carrier replaces one or more nucleotides (plural available) at one or more internal positions (plural available) of the nucleotide sequence of a single-stranded oligonucleotide (e.g., Z 1 and / or Z 2 ).

[0440] As used herein, a ribonucleotide subunit in which the ribose sugar of the subunit is thus replaced is referred to as a ribose-substituted modified subunit (RRMS). The carrier can be a cyclic or acyclic moiety and can include two "skeletal junctions" (e.g., hydroxyl groups) and a ligand. The ligand can be directly conjugated to the carrier or, as described above, indirectly conjugated to the carrier via an intervening linker / tether.

[0441] [Chem.]

[0442] The ligand conjugate monomer subunit may be a 5' or 3' terminal subunit of a single-stranded oligonucleotide sequence (e.g., Z 1 and / or Z 2 ), i.e., one of the two "W" groups may be a hydroxyl group, and the other "W" group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand conjugate monomer subunit may occupy an internal position, and both "W" groups may be one or more unmodified or modified ribonucleotides. More than one ligand conjugate monomer subunit may be present in the single-stranded oligonucleotide.

[0443] Sugar-substituted based monomers, e.g., ligand conjugate monomers (cyclic) Cyclic sugar-substituted based monomers, e.g., sugar-substituted based ligand conjugate monomers are also referred to herein as RRMS monomer compounds. The carrier may have the general formula (LCM-2) provided below (in its structure, preferred backbone junctions are R 1 or R 2 , R 3 or R 4 , or when Y is CR 9 R 10 , R 9 and R 10 may be selected from (the two positions are selected to give two backbone junctions, e.g., R 1 and R 4 , or R 4 and R 9 ). Preferred tethering junctions are when X is CH 2 , R 7 ; R 5 or R 6comprises. The carrier is described below as an entity that can be incorporated with the chain he. Thus, the structure has one (in the case of a terminal position) or two (in the case of an internal position) junction points, for example, R 1 or R 2 R 3 or 4 or R 9 or R 10 (where Y is CR 9 R 10 ) also includes the situation where it is connected to sulfur containing the backbone, such as phosphoric acid or modified phosphoric acid. For example, one of the R groups named above may be -CH 2 -, where one bond is connected to the carrier and one is connected to the backbone atom, for example, connecting an oxygen or a central phosphorus atom.

[0444]

Chemical formula

[0445] Exemplary carriers are, for example, where X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent; or where X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR 11 R 12 ; or where X is N(CO)R 7 or NR 7and Y is O and Z is CR 11 R 12 or X is CH 2 and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form C 6 cycloalkyl (H, z = 2), or an indane ring system, for example, X is CH 2 and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form C 5 cycloalkyl (H, z = 1).

[0446] In certain embodiments, the carrier may be based on a pyrroline ring system or a 4 - hydroxypyrroline ring system, for example, X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is absent (D).

[0447]

Chemical formula

[0448] OFG 1 is preferably joined to a first carbon, for example, an exocyclic alkylene group such as a methylene group, which is attached to one of the carbons of the five - membered ring (the CH of D 2 OFG 1 ). OFG 2 is preferably directly joined to one of the carbons of the five - membered ring (the OFG of D 2 ). In the pyrroline - based carrier, CH 2 OFG 1 may be joined to C - 2, and OFG 2 may be joined to C - 3; or - CH 2 OFG 1may be joined to C-3, OFG 2 may be joined to C-4. In certain embodiments, CH 2 OFG 1 and OFG 2 may be disubstituted on one of the carbons referenced above. In the case of 3-hydroxyproline-based carriers, -CH 2 OFG 1 may be joined to C-2 and OFG 2 may be joined to C-4. Pyrroline- and 4-hydroxyproline-based monomers can thus contain linkages (e.g., carbon-carbon bonds) where bond rotation is restricted by constraints arising from specific linkages, such as the presence of a ring. Thus, CH 2 OFG 1 and OFG 2 can be cis or trans to each other in either of the pairs depicted above. Thus, all cis / trans isomers are explicitly included. The monomer may contain one or more chiral centers and can thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomer are explicitly included (e.g., the centers bearing CH 2 OFG 1 and OFG 2 can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, and vice versa). The tethering junction point is preferably nitrogen. Preferred examples of carrier D are the following:

[0449]

Chemical formula

[0450] In certain embodiments, the carrier may be based on a piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR11 R 12 is.

[0451]

Chemical formula

[0452] In certain embodiments, the carrier may be based on a piperidine ring system (F), e.g., X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or it may be based on a morpholine ring system (G), e.g., X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 is.

[0453]

Chemical Structure

[0454] OFG 1is preferably joined to a first carbon, such as an exocyclic alkylene group, e.g., a methylene group, attached to one of the carbons of the six-membered ring (the -CH of F or G 2 OFG 1 ). OFG 2 is preferably directly joined to one of the carbons of the six-membered ring (the OFG of F or G 2 ). In both F and G, -CH 2 OFG 1 may be joined to C-2 and OFG 2 may be joined to C-3; or vice versa. In certain embodiments, CH 2 OFG 1 and OFG 2 may be disubstituted on one of the carbons referenced above. Piperazine- and morpholine-based monomers can thus contain linkages (e.g., carbon-carbon bonds) where bond rotation is restricted by constraints arising from a particular linkage, e.g., the presence of a ring. Thus, CH 2 OFG 1 and OFG 2 can be cis or trans to each other in either of the pairs described above. Thus, all cis / trans isomers are explicitly included. The monomers may contain one or more chiral centers and can thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomers are explicitly included (e.g., the centers bearing CH 2 OFG 1 and OFG 2 can both have the R configuration; or both can have the S configuration; or one center can have the R configuration and the other center can have the S configuration, and vice versa). The tethering junction points are preferably nitrogen in both F and G.

[0455] In certain embodiments, the carrier may be based on a decalin ring system, e.g., X is CH 2 and Y is CR 9 R 10 and Z is CR 11R 12 is, and R 5 and R 11 both form C 6 cycloalkyl (H, z = 2), or may be based on an indane ring system. For example, X is CH 2 and Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 both form C 5 cycloalkyl (H, z = 1).

[0456]

Chemical Structure

[0457] OFG 1 is preferably bonded to a primary carbon, such as an exomethylene group (n = 1) or an ethylene group (n = 2), connected to one of C-2, C-3, C-4, or C-5 [H of -(CH 2 ) n OFG 1 )]. OFG 2 is preferably directly bonded to one of C-2, C-3, C-4, or C-5 (H of -OFG 2 ). -CH 2 OFG 1 and OFG 2 may be arranged in a paired manner with respect to the ring, i.e., both groups may be bonded to the same carbon, such as C-2, C-3, C-4, or C-5. Alternatively, -(CH 2 ) n OFG 1 and OFG 2 may be arranged in an adjacent manner with respect to the ring, i.e., both groups may be bonded to adjacent ring carbon atoms, such as -(CH 2 ) n OFG 1 may be bonded to C-2 and OFG 2 may be bonded to C-3; -(CH 2 ) n OFG1 may be joined to C-3, OFG 2 may be joined to C-2; -(CH 2 ) n OFG 1 may be joined to C-3, OFG 2 may be joined to C-4; or, -(CH 2 ) n OFG 1 may be joined to C-4, OFG 2 may be joined to C-3; -(CH 2 ) n OFG 1 may be joined to C-4, OFG 2 may be joined to C-5; or -(CH 2 ) n OFG 1 may be joined to C-5, OFG 2 may be joined to C-4. The decalin or indane-based monomer may thus contain linkages where bond rotation is restricted by constraints arising from certain linkages, such as the presence of a ring (e.g., a carbon-carbon bond). Thus, -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans to each other in any of the pairs depicted above. Thus, all cis / trans isomers are explicitly included. The monomer may contain one or more chiral centers and may thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of the monomer are explicitly included (e.g., the centers having 2 OFG 1 and OFG 2 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 to each other. The tethering junction point is preferably C-6 or C-7.

[0458] Other carriers may include those based on 3-hydroxyproline (J).

[0459]

Chemical formula

[0460] Details regarding more representative cyclic, sugar-substituted-based carriers can be found in U.S. Patent Nos. 7,745,608 and 8,017,762, which are hereby incorporated by reference in their entirety.

[0461] Sugar-substituted-based monomer (acyclic) Acyclic sugar-substituted-based monomers, such as sugar-substituted-based ligand conjugate monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers have the formula LCM-3 or LCM-4:

[0462]

Chemical formula

[0463] In some embodiments, each x, y, and z can independently be 0, 1, 2, or 3. In Formula LCM-3, if y and z are different, then the third carbon can have either an R or S configuration. In a preferred embodiment, x is zero, y and z are each 1 in Formula LCM-3 (e.g., based on serinol), and y and z are each 1 in Formula LCM-3. Each of the following Formulas LCM-3 or LCM-4 can be substituted, for example, by hydroxy, alkoxy, perhaloalkyl.

[0464] Details regarding more representative acyclic, sugar-substituted based carriers can be found in U.S. Patent Nos. 7,745,608 and 8,017,762, which are hereby incorporated by reference in their entirety.

[0465] In some embodiments, a single-stranded oligonucleotide comprises one or more ligands conjugated to the 5' end of a nucleotide sequence (e.g., Z 1 and / or Z 2 ).

[0466] In certain embodiments, the ligand is conjugated to the 5' end of a nucleotide sequence (e.g., Z 1 and / or Z 2 ) via a carrier and / or a linker. In one embodiment, the ligand has the formula:

[0467]

Chemical formula

[0468] In some embodiments, a single-stranded oligonucleotide comprises one or more ligands conjugated to the 3' end of a nucleotide sequence (e.g., Z 1 and / or Z 2 ).

[0469] In certain embodiments, the ligand is conjugated to the 3' end of a nucleotide sequence (e.g., Z 1 and / or Z 2 ) via a carrier and / or a linker. In one embodiment, the ligand has the formula:

[0470]

Chemical formula

[0471] In some embodiments, the ligand is conjugated to a nucleotide sequence (e.g., Z 1 and / or Z 2 ) via one or more linkers (tethers) and / or a carrier. In one embodiment, the ligand is conjugated to a nucleotide sequence (e.g., Z 1 and / or Z 2 ) via one or more linkers (tethers).

[0472] In one embodiment, the ligand is conjugated to the 5' or 3' end of a nucleotide sequence (e.g., Z 1 and / or Z 2 ) via a cyclic carrier and optionally one or more intervening linkers (tethers).

[0473] In some embodiments, the ligand is conjugated to at least one nucleotide sequence (e.g., Z 1 and / or Z 2) is conjugated to one or more internal positions thereon. An internal position of a nucleotide sequence refers to a nucleotide at any position of the nucleotide sequence, excluding the terminal positions from the 3'-end and 5'-end of the nucleotide sequence (e.g., position 2: excluding position 1 counted from the 3'-end and position 1 counted from the 5'-end).

[0474] In one embodiment, the ligand is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ), including all positions except the terminal 2 positions from each end of the nucleotide sequence (e.g., position 4: excluding position 1 and position 2 counted from the 3'-end and position 1 and position 2 counted from the 5'-end). In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ), including all positions except the terminal 3 positions from each end of the nucleotide sequence (e.g., position 6: excluding position 1, position 2, and position 3 counted from the 3'-end and position 1, position 2, and position 3 counted from the 5'-end).

[0475] In one embodiment, the ligand is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ), excluding the cleavage site region of the nucleotide sequence. For example, the ligand is not conjugated to positions 9 - 12 counted from the 5'-end of the nucleotide sequence. For example, the ligand is not conjugated to positions 9 - 11 counted from the 5'-end of the nucleotide sequence (e.g., Z 1 and / or Z 2 ). Alternatively, the internal positions exclude positions 11 - 13 counted from the 3'-end of the nucleotide sequence (e.g., Z 1 and / or Z 2 ). In one embodiment, the internal positions exclude positions 12 - 14 counted from the 5'-end of the nucleotide sequence.

[0476] In one embodiment, the ligand is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ), excluding positions 11 to 13 of the nucleotide sequence when counting from the 3' end and positions 12 to 14 of the nucleotide sequence (e.g., Z 1 and / or Z 2 ) when counting from the 5' end.

[0477] In one embodiment, one or more ligands are conjugated to one or more of the following internal positions: positions 4 to 8 and 13 to 18 of the nucleotide sequence (e.g., Z 1 and / or Z 2 ) when counting from the 5' end, and positions 6 to 10 and 15 to 18 of the nucleotide sequence (e.g., Z 1 and / or Z 2 ).

[0478] In one embodiment, one or more ligands are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 of the nucleotide sequence (e.g., Z 1 and / or Z 2 ) when counting from the 5' end, and positions 15 and 17 of the nucleotide sequence (e.g., Z 1 and / or Z 2 ).

[0479] In some embodiments, the ligand is conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a single-stranded oligonucleotide. Ligand

[0480] In certain embodiments, the single-stranded oligonucleotide is further modified by covalent attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the conjugated single-stranded oligonucleotide, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, intracellular distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in the art of chemistry and are linked directly to the parent compound, such as an oligomeric compound, or via any linker moiety or linking group. A preferred list of conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholate moieties, folic acid, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, dyes.

[0481] In some embodiments, the single-stranded oligonucleotide further comprises a targeting ligand that targets a receptor that mediates delivery to a particular CNS tissue. These targeting ligands can be conjugated in combination with a lipophilic moiety to enable specific local (e.g., intrathecal) and systemic delivery.

[0482] Exemplary targeting ligands that target receptor-mediated delivery to CNS tissue are peptide ligands such as Angiopep-2, lipoprotein receptor-related protein (LRP) ligands, bEnd.3 cell-binding ligands; transferrin receptor (TfR) ligands (which can utilize the iron transport system in the brain and cargo transport to the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells, glial cells), glucose transporter proteins, and LDL receptor ligands.

[0483] In some embodiments, the single-stranded oligonucleotide further comprises a targeting ligand that targets a receptor that mediates delivery to a particular eye tissue. These targeting ligands can 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 eye tissue 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 eye endothelial cells).

[0484] Preferred conjugate groups suitable for the present invention include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocolesterol (Oberhauser et al., Nucl. 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 such as di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamine chains or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine moieties or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., including 1996, 277, 923).

[0485] Generally, various entities, such as ligands, can be coupled to the oligomeric compounds described herein. The ligand can include naturally occurring molecules, or recombinant or synthetic molecules. Examples of ligands are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazene, polyethyleneimine, cationic group, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyamino acid, transferrin, bisphosphonate, polyglutamate, polyaspartate, aptamer, asialofetuin, hyaluronic acid, procollagen, immunoglobulin (e.g., antibody), insulin, transferrin, albumin, sugar-albumin conjugate, intercalator (e.g., acridine), crosslinking agent (e.g., psoralen, mitomycin C), porphyrin (e.g., TPPC4, texaphyrin, sapphyrin), heterocyclic aromatic hydrocarbon (e.g., phenazine, dihydrophenazine), artificial endonuclease (e.g., EDTA), lipophilic molecule [e.g., steroid, bile acid, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl) chenodeoxycholic acid, dimethoxytrityl, or phenoxazine], peptide (e.g., alpha-helix type peptide, amphiphilic peptide, RGD peptide, cell permeable peptide, endosome-lysing / membrane-fusing peptide), alkylating agent, phosphoric acid, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (e.g., biotin), transport / absorption promoter (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ complex of tetraaza macrocycle), dinitrophenyl, HRP, AP, antibody, hormone and hormone receptor, lectin, carbohydrate, polyvalent carbohydrate, vitamin (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactor, lipopolysaccharide, activator of p38MAP kinase, activator of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNF alpha), interleukin-1 beta, gamma interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high density lipoprotein (HDL), and cell permeating agent (e.g., helix type cell permeating agent) including but not limited to these.,

[0486] Peptides and peptidomimetic ligands include naturally occurring or modified peptides, such as D- or L-peptides; α-, β-, or γ-peptides; N-methyl peptides; azapeptides; peptides in which one or more amides, i.e., peptide bonds, are replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to that of a natural peptide. A peptide or peptidomimetic ligand can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0487] Exemplary amphiphilic peptides include cecropin, ricin toxin, pardaxin, buforin, CPF, bomvinin-like peptide (BLP), cathelicidin, serratotoxin, cecropin, ricin toxin, pardaxin, buforin, CPF, bomvinin-like peptide (BLP), cathelicidin, serratotoxin, S. clava peptide, HFIAP, magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H 2 A peptide, Xenopus peptide, esculentinis-1, caerin, and the like, but not limited thereto.

[0488] As used herein, the term "endosomolytic ligand" refers to a molecule having endosomolytic properties. An endosomolytic ligand promotes the lysis of the compositions of the present invention, or its components, and / or the transport from cell compartments such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other vesicles within the cell to the cytoplasm. Some exemplary endosomolytic ligands include, but are not limited to, imidazole, poly or oligoimidazole, linear or branched polyethyleneimine (PEI), linear and branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers having masked or unmasked cationic or anionic charges, dendrimers having masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptide mimetics, pH-sensitive peptides, natural and synthetic membrane-fusion lipids, and natural and synthetic cationic lipids.

[0489] Exemplary endosome-lysing / membrane-fusing peptides include, but are not limited to, AALEALAEALEALAEALEALAEAAAAGGC(GALA) (SEQ ID NO: 3); AALAEALAEALAEALAEALAEALAAAAGGC(EALA) (SEQ ID NO: 4); ALEALAEALEALAEA (SEQ ID NO: 5); GLFEAIEGFIENGWEGMIWDYG(INF-7) (SEQ ID NO: 6); GLFGAIAGFIENGWEGMIDGWYG(Inf HA-2) (SEQ ID NO: 7); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMIDGWYGC(diINF-7) (SEQ ID NO: 8); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC(diINF-3) (SEQ ID NO: 9); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC(GLF) (SEQ ID NO: 10); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC(GALA-INF3) (SEQ ID NO: 11); GLFEAIEGFIENGWEGnIDGKGLFEAIEGFIENGWEGnIDG(INF-5, n is norleucine) (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(merittin) (SEQ ID NO: 18); H 5 WYG (SEQ ID NO: 19); and CHK 6 HC (SEQ ID NO: 20), among others.

[0490] Speaking without wishing to be bound by theory, membrane-fusing lipids fuse with the membrane and as a result destabilize the membrane. Membrane-fusing lipids typically have a small head group and unsaturated acyl chains. Exemplary membrane-fusing lipids include, but are not limited to, 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanolamine (also referred to herein as XTC).

[0491] Synthetic polymers having endosomal-lysing activity suitable for the present invention are described in U.S. Patent Application Publication Nos. 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.

[0492] Exemplary cell-penetrating peptides include, but are not limited to, RQIKIWFQNRRMKWKK (penetratin) (SEQ ID NO: 21); GRKKRRQRRRPPQC (Tat fragment 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: 29); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO: 30); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO: 31); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO: 32); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID NO: 33); ILPWKWPWWPWRR-NH2 (indolicidin) (SEQ ID NO: 34); AAVALLPAVLLALLAP (RFGF) (SEQ ID NO: 35); AALLPVLLAAP (RFGF analog) (SEQ ID NO: 36); and RKCRIVVIRVCR (bactenecin) (SEQ ID NO: 37).

[0493] Exemplary cationic groups include, for example, O-AMINE (AMINE = NH 2 ; protonated amino groups derived from alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, for example, O(CH 2 ) n AMINE (AMINE = NH 2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE (AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino) and is not limited thereto.

[0494] As used herein, the term "targeting ligand" refers to any molecule that enhances the affinity for a selected target, such as a cell, cell type, tissue, organ, region within 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, 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.

[0495] Carbohydrate-based targeting ligands include D-galactose, polyvalent galactose, N-acetyl-D-galactosamine (GalNAc), polyvalent GalNAc, e.g., GalNAc 2 , and GalNAc 3(GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); including, but not limited to, D-mannose, multivalent mannose, multivalent lactose, N-acetylglucosamine, glucose, multivalent glucose, multivalent fucose, glycosylated polyamino acids, and lectins. The term multivalent indicates the presence of more than one monosaccharide unit. Such monosaccharide subunits can be linked to each other via glycosidic linkages or to a scaffold molecule.

[0496] A number of folic acids and folic acid analogs suitable for use in the present invention as ligands are described in U.S. Patent Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the contents of which are incorporated herein by reference in their entirety.

[0497] As used herein, the terms "PK modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of the compositions of the present invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and trans-thyretin binding ligands (e.g., tetraiidothyroacetic acid, 2,4,6-triiodophenol and flufenamic acid). Oligomeric compounds containing multiple phosphorothioate sugar linkages are also known to bind to serum proteins, and thus short oligomeric 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 the backbone are also suitable for the present invention as ligands (e.g., as PK modulating ligands). PK modulating oligonucleotides can 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 for the present invention as PK modulating ligands. Binding to serum components (e.g., serum proteins) can be predicted from albumin binding assays as described in Oravcova, et al., Journal of Chromatography B (1996), 677: 1-27.

[0498] 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 other ligands have different properties. For example, a ligand may have targeting properties, endosomal lysis activity, or PK modulating properties. In a preferred embodiment, all ligands have different properties.

[0499] A ligand or tethered ligand may be present on the monomer when the monomer is incorporated into the component of the single-stranded oligonucleotide. In some embodiments, the ligand may be incorporated via coupling to the "precursor" monomer after the "precursor" monomer is incorporated into the component of the single-stranded oligonucleotide. For example, a monomer having, for example, an amino-terminal tether (i.e., having no associated ligand), for example, monomer-linker-NH 2 can be incorporated into the component of the single-stranded oligonucleotide. In a subsequent operation, i.e., after the precursor monomer is incorporated into the component of the single-stranded oligonucleotide, a ligand having an electrophilic group, for example, a pentafluorophenyl ester or an aldehyde group, can be subsequently conjugated to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the tether of the precursor monomer.

[0500] In another example, a monomer having a chemical group suitable for participating in a click chemistry reaction, for example, an azide or alkyne-terminated tether / linker, can be incorporated. In a subsequent operation, i.e., after the precursor monomer is incorporated into the chain, a ligand having a complementary chemical group, for example, an alkyne or azide, can be conjugated to the precursor monomer by coupling the alkyne and azide together.

[0501] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a single-stranded oligonucleotide. Conjugation to a purine nucleobase or its derivative can occur at any position including atoms within the ring and atoms outside the ring. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is joined to the conjugate moiety. Conjugation to a pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be substituted with the conjugate moiety. When the ligand is conjugated to the nucleobase, the preferred positions are those that do not interfere with hybridization, i.e., those that do not interfere with the hydrogen bonding interactions necessary for base pair formation.

[0502] Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be joined to the conjugate moiety include the 2’, 3’, and 5’ carbon atoms. The 1’ position can also be joined to the conjugate moiety, such as within an abasic residue. The internucleoside linkage can also carry a conjugate moiety. In the case of a phosphorus-containing linkage (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be directly joined to the phosphorus atom or to an O, N, or S atom bonded to the phosphorus atom. In the case of an amine or amide-containing internucleoside linkage (e.g., PNA), the conjugate moiety can be joined to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0503] There are numerous methods for preparing conjugates of oligonucleotides. Generally, the oligonucleotide is joined at the 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 moiety. In some embodiments, one reactive group is electrophilic and the other reactive group is nucleophilic.

[0504] For example, the electron-withdrawing group can be a carbonyl-containing functionality and the nucleophilic group can be an amine or a thiol. Methods of conjugation of nucleic acids and related oligomeric compounds with or without a linking group are well described in the literature such as, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.

[0505] Representative U.S. patents that teach the preparation of nucleic acid conjugates include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031, the contents of which are incorporated herein by reference in their entirety.Same as U.S. Patent No. 6,528,631; including, but not limited to, U.S. Patent No. 6,559,279;

[0506] 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.

[0507] In some embodiments, the carbohydrate-based ligand is any one of the ligands listed in Table 2, Table 2A, Table 3, Table 3A, Table 4, or Table 4A of WO2015 / 006740, which is hereby incorporated by reference in its entirety.

[0508] In some embodiments, the linker, such as a branched linker that is a divalent or trivalent branched linker that conjugates these carbohydrate-based ligands, comprises the linker(s) listed in Table 1 or Table 1A of WO2015 / 006740 and the spacer(s) listed in Table 5, which are hereby incorporated by reference in their entirety.

[0509] In some embodiments, the GalNAc-based conjugate is a GalNAc analog containing an S or N atom, or a CH 2 group in the glycosidic linkage that changes a metabolically labile glycosidic linkage to a metabolically stable glycosidic linkage, for example, as shown by the following scheme, where the "O" in the glycosidic linkage is replaced by an S or N atom, or a -CH 2 - group.

[0510]

Chemical formula

[0511] In some embodiments, the GalNAc-based conjugate is a GalNAc analog having one of the following structures:

[0512] [Table 2] TIFF2025516680000097.tif240160

[0513] The GalNAc analogs listed in the above table can be prepared using the methods described in WO2015 / 006740, which is incorporated herein by reference in its entirety.

[0514] In some embodiments, the GalNAc-based conjugate has the following structure:

[0515] [Chemical Structure] [where n = 0 to 10 (e.g., 1 or 4). See Figures 4A and 4B of US2021 / 0123048A1, which is incorporated herein by reference in its entirety.]

[0516] [Chemical Structure] is a GalNAc analog having one of the following:

[0517] In certain embodiments, the single-stranded oligonucleotide has the structure shown below:

[0518]

Chemical formula

[0519] In certain embodiments, the single-stranded oligonucleotide is of formula (II), (III), (IV) or (V):

[0520]

Chemical formula

[0521]

Chemical formula

[0522] As discussed above, since the ligand can be conjugated to a single-stranded oligonucleotide via a linker or a carrier, and since the linker or carrier can contain a branched linker, the single-stranded oligonucleotide can contain multiple ligands via the same or different backbone attachment points to the carrier, or via one or more branched linkers. For example, the branching point of the branched linker can be a divalent, trivalent, tetravalent, pentavalent, or hexavalent atom, or a group exhibiting such multiple valences. In certain embodiments, the branching point is -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)O-C, [wherein Q is, independently, for each occurrence, H or an appropriately substituted alkyl]. In other embodiments, the branching point is glycerol or a glycerol derivative.

[0523] In certain embodiments, the ASGPR ligand conjugated to the single-stranded oligonucleotide is one or more GalNAc derivatives joined by a divalent or trivalent branched linker.

[0524] In certain embodiments, the single-stranded oligonucleotide

[0525]

Chemical formula

[0526] In certain embodiments, the single-stranded oligonucleotide

[0527]

Chemical formula

[0528] In certain embodiments, the single-stranded oligonucleotide

[0529]

Chemical formula

[0530] In certain embodiments, the single-stranded oligonucleotide

[0531]

Chemical formula

[0532] In certain embodiments, the single-stranded oligonucleotide

[0533]

Chemical formula

[0534] In certain embodiments, the single-stranded oligonucleotide comprises

[0535]

Chemical formula

[0536] In certain embodiments, the single-stranded oligonucleotide comprises

[0537]

Chemical formula

[0538] In certain embodiments, the single-stranded oligonucleotide comprises

[0539]

Chemical formula

[0540] In certain embodiments, the single-stranded oligonucleotide comprises

[0541]

Chemical formula

[0542] In certain embodiments, the single-stranded oligonucleotide comprises

[0543]

Chemical formula

[0544] In certain embodiments, the single-stranded oligonucleotide comprises

[0545]

Chemical formula

[0546] In certain embodiments, the single-stranded oligonucleotide

[0547]

Chemical formula

[0548] In certain embodiments, the single-stranded oligonucleotide

[0549]

Chemical formula

[0550] In certain embodiments, the single-stranded oligonucleotide

[0551]

Chemical formula

[0552] In certain embodiments, the single-stranded oligonucleotide

[0553]

Chemical formula

[0554] In certain embodiments, the single-stranded oligonucleotide

[0555]

Chemical formula

[0556] In certain embodiments, the single-stranded oligonucleotide

[0557]

Chemical formula

[0558] In certain embodiments, the single-stranded oligonucleotide

[0559]

Chemical formula

[0560] In certain embodiments, the single-stranded oligonucleotide

[0561]

Chemical formula

[0562] In certain embodiments, the single-stranded oligonucleotide

[0563]

Chemical formula

[0564] In certain embodiments, the single-stranded oligonucleotide

[0565]

Chemical formula

[0566] In certain embodiments, the single-stranded oligonucleotide

[0567]

Chemical formula

[0568] In certain embodiments, the single-stranded oligonucleotide

[0569] [Chemical formula] contains the ligand of

[0570] In certain embodiments, the single-stranded oligonucleotide is

[0571] [Chemical formula] contains the ligand of

[0572] In certain embodiments, the single-stranded oligonucleotide is

[0573] [Chemical formula] contains the ligand of

[0574] In certain embodiments, the single-stranded oligonucleotide is

[0575] [Chemical formula] contains the ligand of

[0576] In certain embodiments, the single-stranded oligonucleotide is

[0577] [Chemical formula] contains the ligand of

[0578] In certain embodiments, the single-stranded oligonucleotide is

[0579] [Chemical formula] contains the ligand of

[0580] In certain embodiments, the single-stranded oligonucleotide is

[0581]

Chem.

[0582] In certain embodiments, the single-stranded oligonucleotide is

[0583]

Chem.

[0584] In certain embodiments, the single-stranded oligonucleotide is

[0585]

Chem.

[0586] In certain embodiments, the single-stranded oligonucleotide is

[0587]

Chem.

[0588] In certain embodiments, the single-stranded oligonucleotide of the present invention is

[0589]

Chem.

[0590] In certain embodiments, the single-stranded oligonucleotide is

[0591]

Chem.

[0592] In some embodiments, L 2Aand L 2B are both the same. In some embodiments, L 2A and L 2B are both different.

[0593] In some embodiments, L 3A and L 3B are both the same. In some embodiments, L 3A and L 3B are both different.

[0594] In some embodiments, L 4A and L 4B are both the same. In some embodiments, L 4A and L 4B are both different.

[0595] In some embodiments, L 5A , L 5B and L 5C are all the same. In some embodiments, L 5A , L 5B and L 5C two of them are the same. In some embodiments, L 5A and L 5B are the same. In some embodiments, L 5A and L 5C are the same. In some embodiments, L 5B and L 5C are the same.

[0596] In certain embodiments, the single-stranded oligonucleotide comprises

[0597]

Chemical formula

[0598] In certain embodiments, the single-stranded oligonucleotide comprises

[0599]

Chemical formula

[0600] In certain embodiments, the single-stranded oligonucleotide

[0601]

Chemical formula

[0602] In certain embodiments, the single-stranded oligonucleotide

[0603]

Chemical formula

[0604] In certain embodiments, the single-stranded oligonucleotide

[0605]

Chemical formula

[0606] In certain embodiments, the single-stranded oligonucleotide

[0607]

Chemical formula

[0608] In certain embodiments, the single-stranded oligonucleotide has the structure:

[0609]

Chemical formula

[0610] In some embodiments, the single-stranded oligonucleotide

[0611]

Chemical formula

[0612] In some embodiments, the single-stranded oligonucleotide

[0613]

Chemical formula

[0614] In some embodiments, the single-stranded oligonucleotide

[0615]

Chemical formula

[0616] In some embodiments, the single-stranded oligonucleotide

[0617]

Chemical formula

[0618] In certain embodiments, the single-stranded oligonucleotide

[0619]

Chemical formula

[0620] In certain embodiments, the single-stranded oligonucleotide is

[0621]

Chemical formula

[0622] In certain embodiments, the single-stranded oligonucleotide is

[0623]

Chemical formula

[0624] In certain embodiments, the single-stranded oligonucleotide is

[0625]

Chemical formula

[0626] In some embodiments, the single-stranded oligonucleotide is

[0627]

Chemical formula

[0628] In some embodiments, the single-stranded oligonucleotide is

[0629]

Chemical formula

[0630] In some embodiments, the single-stranded oligonucleotide is

[0631] [Chemical formula] [wherein, R is OH or NHCOCH 3 is] and contains monomers of

[0632] In certain embodiments, the single-stranded oligonucleotide is

[0633] [Chemical formula] [wherein, R is OH or NHCOCH 3 is] and contains monomers of

[0634] In certain embodiments, the single-stranded oligonucleotide is

[0635] [Chemical formula] and contains monomers of

[0636] In the above monomers, X and Y are each 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, -P(Z’)(Z’’)O-nucleoside, -P(Z’)(Z’’)O-oligonucleotide, a lipid, PEG, a steroid, a polymer, a nucleotide, a nucleoside, or an oligonucleotide; and Z’ and Z’’ are each independently, for each occurrence, O or S.

[0637] In some embodiments, the single-stranded oligonucleotide is

[0638] [Chem.] is conjugated to a ligand of

[0639] In certain embodiments, the single-stranded oligonucleotide

[0640] [Chem.] contains a ligand of

[0641] In certain embodiments, the single-stranded oligonucleotide

[0642] [Chem.] contains a monomer of . The synthesis of the above ligands and monomers is described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference.

[0643] In certain embodiments, at least one ligand conjugated to the single-stranded oligonucleotide is a lipophilic moiety.

[0644] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, logK ow by, K owis the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a characteristic measured in the laboratory for a substance. However, it can also be predicted by using coefficients attributed to the structural components of the chemical substance, calculated using first principles or experimental methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measure of the tendency of a substance to prefer a non-aqueous or oily environment over water (i.e., its hydrophilic / lipophilic balance). In principle, for a chemical substance, if its logK ow exceeds 0, the property is lipophilic. Typically, lipophilic moieties have a logK ow greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. For example, the logK ow of 6-aminohexanol is predicted to be, for example, approximately 0.7. Using the same method, the logK ow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.

[0645] The lipophilicity of a molecule can be altered with respect to the functional groups it bears. For example, the addition of a hydroxyl group or an amine group to the terminus of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logK ow ) value of the lipophilic moiety.

[0646] 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 correlate positively with the relative hydrophobicity of the single-stranded oligonucleotide, which can correlate positively with the silencing activity of the single-stranded oligonucleotide.

[0647] In one embodiment, the determined plasma protein binding assay is an electrophoretic mobility shift assay (EMSA) that uses human serum albumin protein. The hydrophobicity of the single-stranded oligonucleotide, as measured by the fraction of unbound single-stranded oligonucleotide 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 enhancing the in vivo delivery of the single-stranded oligonucleotide.

[0648] Accordingly, conjugation of the lipophilic moiety to the internal position(s) of the single-stranded oligonucleotide provides an optimal hydrophobicity for enhancing the in vivo delivery of the single-stranded oligonucleotide.

[0649] In certain embodiments, the lipophilic moiety is an aliphatic, cyclic such as alicyclic, or polycyclic such as polycyclic alicyclic compound, e.g., a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon. The lipophilic moiety generally includes a hydrocarbon chain that can be cyclic or acyclic. The hydrocarbon chain can include various substituents and / or one or more heteroatoms, such as an oxygen atom or a nitrogen atom. Such lipophilic aliphatic moieties include saturated or unsaturated C 4 ~C 30 hydrocarbon chains (e.g., C 6 ~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 tetraterpenes) and other polycyclic alicyclic hydrocarbons, but are not limited thereto. For example, the lipophilic moiety is a C 4 ~C 30 hydrocarbon chain (e.g., C 4 ~C 30may contain (alkyl or alkenyl). In some embodiments, the lipophilic moiety is a saturated or unsaturated C 6 ~C 18 hydrocarbon chain (e.g., linear C 6 ~C 18 alkyl or alkenyl) or a saturated or unsaturated C 14 ~C 24 hydrocarbon chain (e.g., linear C 14 ~C 24 alkyl or alkenyl). In one embodiment, the lipophilic moiety is a saturated or unsaturated C 16 hydrocarbon chain (e.g., linear C 16 alkyl or alkenyl) or a saturated or unsaturated C 22 hydrocarbon chain (e.g., linear C 22 alkyl or alkenyl).

[0650] The lipophilic moiety can be conjugated 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 hydroxy group (e.g., -CO-CH 2 -OH). Functional groups already present in the lipophilic moiety or introduced into the single-stranded oligonucleotide include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne.

[0651] Conjugation of the single-stranded oligonucleotide and the lipophilic moiety can occur, for example, by formation of an ether or carboxylic acid or carbamoyl ester linkage between a hydroxy 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 a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group, etc.

[0652] In some embodiments, the lipophilic moiety is conjugated to a single-stranded oligonucleotide via a linker, and the linker contains an ether, thioether, urea, carboxylic acid, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0653] In another embodiment, the lipophilic moiety is a steroid such as a sterol. A 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, digitoxigenin, testosterone, cholesterol, and cationic steroids such as cortisone. A "cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition, or removal of a substituent.

[0654] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" broadly refers to monocyclic and polycyclic aromatic hydrocarbons. Aromatic groups include C 6 ~C 14 aryl moieties; "aralkyl" or "arylalkyl" groups containing an aryl group covalently bonded to an alkyl group, wherein any of which may independently be optionally substituted or unsubstituted; and "heteroaryl" groups, among others. 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 14 π electrons shared in a cyclic array; and having between 1 and about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S) in addition to carbon atoms.

[0655] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group has from 1 to about 4, preferably from 1 to about 3, more preferably 1 or 2 non-hydrogen substitutions. Suitable substitutions include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0656] In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected such that the lipophilic moiety binds to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected such that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group facilitate binding to albumin, immunoglobulin, lipoprotein, α-2-macroglobulin, or α-1-glycoprotein.

[0657] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Synthetic procedures for naproxen can be found in U.S. Patent No. 3,904,682 and U.S. Patent No. 4,009,197, which are hereby incorporated by reference in their entirety. Naproxen has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid and the structure is

[0658]

Chemical Structure

[0659] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. The synthetic procedure for ibuprofen can be found in U.S. Patent No. 3,228,831, which is hereby incorporated by reference in its entirety. The structure of ibuprofen is

[0660]

Chemical Formula

[0661] Further exemplary aralkyl groups are exemplified in U.S. Patent No. 7,626,014, which is hereby incorporated by reference in its entirety.

[0662] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 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.

[0663] In some embodiments, the lipophilic moiety is a C 6 ~C 30 acid (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-docosahexaenoic acid, vitamin A, vitamin E, cholesterol, etc.) or a C 6 ~C 30It is alcohol (such as 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 moiety is docosahexaenoic acid.

[0664] In certain embodiments, particularly when the lipophilic moiety has low lipophilicity or hydrophobicity, more than one lipophilic moiety can be incorporated into the single-stranded oligonucleotide. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of the single-stranded oligonucleotide. In one embodiment, each strand of the single-stranded oligonucleotide has one or more incorporated lipophilic moieties. In one embodiment, two or more lipophilic moieties are incorporated at 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 that continuously link the lipophilic moieties.

[0665] The lipophilic moiety can be conjugated to the single-stranded oligonucleotide via direct conjugation to the ribose of the single-stranded oligonucleotide. Alternatively, the lipophilic moiety can be conjugated to the single-stranded oligonucleotide via a linker or a carrier.

[0666] In certain embodiments, the lipophilic moiety can be conjugated to the single-stranded oligonucleotide via one or more linkers (tethers).

[0667] In one embodiment, the lipophilic moiety is conjugated to the single-stranded oligonucleotide via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, a product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0668] Definitions Unless otherwise specified, the nomenclature, as well as the procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein, are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Certain such techniques and procedures are found, for example, in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference in their entirety for all purposes. All patents, applications, published applications, and other publications and other data referred to throughout this disclosure, if permitted, are hereby incorporated by reference in their entirety.

[0669] Unless otherwise indicated, the following terms have the following meanings.

[0670] 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 derived from such RNA, and miRNA. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state. In some embodiments, the target nucleic acid can be a nucleic acid molecule derived from an infectious pathogen.

[0671] As used herein, the term "iRNA" refers to an agent that mediates targeted cleavage of an RNA transcript. These agents associate with a cytoplasmic multi-protein complex known as the RNAi-induced silencing complex (RISC). Agents effective to induce RNA interference are also referred to herein as siRNA, RNAi agents, or iRNA agents. Thus, these terms can be used interchangeably herein. As used herein, the term iRNA includes microRNA and pre-microRNA. Further, the "compound" or "compounds" of the invention as used herein also refers to an iRNA agent and can be used interchangeably with an iRNA agent.

[0672] The iRNA agent must contain a region having sufficient homology to the target gene, have a sufficient length with respect to nucleotides, and be capable of mediating down-regulation of the target gene by the iRNA agent or a fragment thereof. (For ease of explanation, in this specification, the terms "nucleotide" or "ribonucleotide" may be used with respect to one or more monomeric subunits of the iRNA agent. It will be understood that in this specification, the usage of the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or substitute replacement moieties at one or more positions in the case of modified RNA or nucleotide surrogates). Thus, the iRNA agent is at least partially, and in some embodiments completely, complementary to or contains a region complementary to the target RNA. Complete complementarity between the iRNA agent and the target is not required, but the iRNA agent or its cleavage product must have sufficient complementarity to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA, e.g., mRNA. The degree of complementarity, or homology to the target strand, is most important in the antisense strand. Complete complementarity is often desired, particularly in the antisense strand, but some embodiments may contain one or more, e.g., 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA), particularly in the antisense strand. The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded nature of the molecule.

[0673] An iRNA agent includes a molecule that is long enough to cause an interferon response [cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and capable of entering RISC (RNAi-induced silencing complex)], and a molecule that is short enough not to cause an interferon response (this molecule can also be cleaved by Dicer and / or enter RISC), for example, a molecule of a size capable of entering RISC, such as a molecule similar to a cleavage product by Dicer. A molecule short enough not to cause an interferon response is referred to herein as an siRNA agent or a shorter iRNA agent. As used herein, "siRNA agent or shorter iRNA agent" refers to an iRNA agent that is short enough not to induce a harmful interferon response in human cells, for example, a double-stranded RNA agent or a single-stranded agent, for example, having a double-stranded region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or a cleavage product thereof, can downregulate a target gene, for example, by inducing RNAi with respect to the target RNA, and the target may include endogenous or pathogen target RNA.

[0674] As used herein, "single-stranded oligonucleotide" or "single-stranded iRNA agent" refers to an oligonucleotide or iRNA agent composed of one molecule. The single-stranded oligonucleotide may contain a double-stranded region formed by intrastrand pairing, and may be, for example, a hairpin structure, a dumbbell structure, or a panhandle structure, or may contain them. The single-stranded oligonucleotide or iRNA agent may be antisense to a target molecule. The single-stranded oligonucleotide or iRNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of the target mRNA. The single-stranded oligonucleotide or iRNA agent has a length of at least 14 nucleotides, and in other embodiments, at least 15, 20, 25, 29, 35, 40, or 50 nucleotides. In certain embodiments, the length is less than 200, 100, or 60 nucleotides. In certain embodiments, the single-stranded oligonucleotide contains two oligonucleotides linked by a linking group.

[0675] A loop refers to a region in which a part of an oligonucleotide or iRNA strand does not pair with the opposing nucleotides in the double-strand when forming a base pair with another strand or another part of the same strand.

[0676] The hairpin oligonucleotide or iRNA agent will 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 have a length of 200, 100, or 50 or less. In certain embodiments, the double-stranded region ranges in length from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs. The hairpin may have a single-stranded overhang or terminal unpaired region at the 3' end in some embodiments, and at the antisense side of the hairpin in certain embodiments. In some embodiments, the overhang has a length of 2 to 3 nucleotides.

[0677] As used herein, the terms "double-stranded oligonucleotide", "double-stranded nucleic acid agent", or "double-stranded (ds)iRNA agent" refer to an oligonucleotide, nucleic acid agent, or iRNA agent comprising one or more, and in some cases two strands, capable of intermolecular hybridization to form a region of double-stranded structure.

[0678] As used herein, the terms "activity", "siRNA activity", or "RNAi activity" refer to gene silencing by an oligonucleotide, nucleic acid agent, or iRNA agent.

[0679] As used herein, "gene silencing" by an oligonucleotide, nucleic acid agent, or iRNA agent refers to a decrease in the mRNA level in a cell having a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to 100%, and any integer between the mRNA level found in the cell in the absence of miRNA or RNA interference molecules. In a preferred embodiment, the mRNA level decreases by 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%.

[0680] 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 such that it is greater or less than the expression, level, or activity observed in the absence of the modulator. For example, the term "modulate" means "inhibit", but the use of the term "modulate" is not limited to this definition.

[0681] As used herein, gene expression modulation occurs when the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more different from that observed in the absence of siRNA. The % and / or fold difference is calculated, for example,

[0682]

Number

[0683]

Number

[0684] As used herein, the terms "inhibit", "down-regulate", or "reduce" in relation to gene expression mean that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced compared to that observed in the absence of the modulator. Gene expression is down-regulated when the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced by at least 10% compared to the corresponding non-modulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably 100% (i.e., no gene expression).

[0685] As used herein, the terms "increased" or "upregulated" in relation to gene expression mean that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased compared to what is observed in the absence of a modulator. Gene expression is upregulated when the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased by at least 10% compared to the corresponding unmodulated control, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more.

[0686] As used herein, the terms "increased" or "increasing" generally mean a statistically significant amount of increase. To avoid doubt, "increased" means an increase of at least 10% compared to a reference level, e.g., 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% increase, or at least about 90%, or up to 100% increase, or any increase between 10 - 100% compared to the reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to the reference level, or any increase between 2-fold and 10-fold or more compared to the reference level.

[0687] As used herein, the terms "reduced" or "reducing" generally mean a statistically significant amount of decrease. However, to avoid ambiguity, "reduced" means a decrease of at least 10% compared to a reference level, for example, 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 non-existent level compared to a reference sample), or any decrease between 10 and 100% compared to a reference level.

[0688] The double-stranded nucleic acid agent comprises two oligonucleotide strands having sufficient complementarity to hybridize and form a double-stranded structure. Generally, the double-stranded structure is a length of base pairs between 8 and 30, between 15 and 30, between 18 and 25, between 19 and 24, or between 19 and 21. In some embodiments, a long double-stranded nucleic acid agent having a length of base pairs between 25 and 30 is preferred. In some embodiments, a short double-stranded nucleic acid agent having a length of base pairs between 10 and 15 is preferred. In another embodiment, the double-stranded nucleic acid agent is at least 21 nucleotides in length.

[0689] As used herein, the phrases "antisense strand" or "antisense oligonucleotide" refer to an oligomeric compound that is substantially or 100% complementary to a target sequence of interest. The phrase "antisense strand" includes the antisense regions of both oligomeric compounds formed from two separate strands and single-molecule oligomeric compounds capable of forming hairpin or dumbbell-shaped structures. The terms "antisense strand" and "guide strand" are used interchangeably herein.

[0690] The phrase "sense strand" refers to an oligomeric compound having a nucleoside sequence that is all or part the same as a target sequence such as a messenger RNA or DNA sequence. The terms "sense strand" and "passenger strand" are used interchangeably herein.

[0691] "Specifically hybridizable" and "complementary" mean that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence either in the conventional Watson-Crick type or other non-conventional type. For the nucleic acid molecules of the present invention, the binding free energy between a nucleic acid molecule and its complementary sequence is sufficient for the related functions of the nucleic acid, such as promoting RNAi activity. The determination of the binding free energy of nucleic acid molecules is well-known in the art (see, for example, Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, / . Am. Chem. Soc. 109:3783-3785). Percent complementarity refers to the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick type base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, 100% complementary). "Perfect complementarity" or 100% complementarity means that all consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues of a second nucleic acid sequence. Less than perfect complementarity refers to a situation where some, but not all, of the nucleoside units of the two strands can hydrogen bond with each other. "Substantial complementarity" refers to a polynucleotide strand showing 90% or more complementarity, excluding regions of the polynucleotide strand selected to be non-complementary, such as overhangs. Specific binding requires a degree of complementarity sufficient to avoid non-specific binding of an oligomeric compound to non-target sequences under the conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0692] In some embodiments, the double-stranded region 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, 24, 25, 26, 27, 28, 29, 30, or more nucleotide pairs in length.

[0693] In some embodiments, the first oligonucleotide of the double-stranded nucleic acid agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0694] 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, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0695] In one embodiment, the first and second oligonucleotides of the double-stranded nucleic acid agent are each 15 to 30 nucleotides in length.

[0696] In one embodiment, the first and second oligonucleotides of the double-stranded nucleic acid agent are each 19 to 25 nucleotides in length.

[0697] In one embodiment, the first and second oligonucleotides of the double-stranded nucleic acid agent are each 21 to 23 nucleotides in length.

[0698] In some embodiments, one oligonucleotide has at least one stretch of 1 to 5 single-stranded nucleotides in the double-stranded region. By "stretch of single-stranded nucleotides in the double-stranded region" is meant that there are at least one nucleotide base pair at both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. When both strands have a stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region, such single-stranded nucleotides can be opposite each other (e.g., a stretch of mismatches), or can be arranged such that the second oligonucleotide does not have single-stranded nucleotides that are opposite the single-stranded nucleotides of the first oligonucleotide, vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are present within 8 nucleotides from either end, e.g., within 8 nucleotides from either the 5' or 3' end of the complementary region between two oligonucleotides, e.g., within 8, 7, 6, 5, 4, 3, or 2 nucleotides.

[0699] In one embodiment, the double-stranded nucleic acid agent comprises a single-stranded overhang at at least one of the ends. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0700] In one embodiment, the second oligonucleotide of the double-stranded nucleic acid agent is 21 nucleotides in length and the first oligonucleotide is 23 nucleotides in length, where the first and second oligonucleotides form a double-stranded region of 21 consecutive base pairs with a single-stranded overhang that is 2 nucleotides longer at the 3' end.

[0701] In some embodiments, each oligonucleotide of the double-stranded nucleic acid agent has a ZXY structure as described in PCT Publication No. WO 2004 / 080406, which is incorporated herein by reference in its entirety.

[0702] In certain embodiments, two nucleotide sequences can be ligated together to form a long chain. The two nucleotide sequences can be ligated together by an oligonucleotide linker, including, but not limited to, (N) n (wherein N is independently a modified or unmodified nucleotide and n is from 3 to 23). In some embodiments, n is from 3 to 10, such as 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 can be involved in base pair interactions with other nucleotides in the linker. The two nucleotide sequences can be ligated together by a non-nucleotide-based linker, such as the linkers described herein. It will be understood by those skilled in the art that any oligonucleotide chemical modification or variation described herein can be used for the oligonucleotide linker.

[0703] In certain embodiments, the two strands specifically hybridize if there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, and the conditions under which the assay is performed in the case of in vitro assays.

[0704] As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense compound hybridizes to its target sequence, but hybridizes to other sequences only to a minimal number. Stringent conditions are sequence-dependent and will be different in different circumstances. The "stringent conditions" under which an antisense compound hybridizes to a target sequence are determined by the nature and composition of the antisense compound, as well as the assay in which they are being studied.

[0705] 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. One of ordinary skill in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, such as by determining the melting temperature (Tm). Tm or ΔTm can be calculated by techniques well known to those of ordinary skill in the art. For example, by the techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443), one of ordinary skill in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.

[0706] A single-stranded oligonucleotide can contain a phosphorus-containing group at the 5' end of the nucleotide sequence. The phosphorus-containing group at the 5' end can be a 5' end phosphate (5'-P), 5' end phosphorothioate (5'-PS), 5' end phosphorodithioate (5'-PS 2 ), 5' end vinylphosphonate (5'-VP), 5' end methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl (

[0707]

Chemical formula

[0708] [Chemical formula] ), the 5'-Z-VP isomer (i.e., cis-vinylphosphonate,

[0709] [Chemical formula] ), or a mixture thereof.

[0710] In one embodiment, the single-stranded oligonucleotide contains a phosphorus-containing group at the 5'-end of a nucleotide sequence (e.g., Z 1 and / or Z 2 ).

[0711] In one embodiment, the single-stranded oligonucleotide contains 5'-P in at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ).

[0712] In one embodiment, the single-stranded oligonucleotide contains 5'-PS in at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ).

[0713] In one embodiment, the single-stranded oligonucleotide contains 5'-VP in at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ). In one embodiment, the single-stranded oligonucleotide contains 5'-A1-VP in at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ). In one embodiment, the single-stranded oligonucleotide contains 5'-A1-VP in at least one nucleotide sequence (e.g., Z 1 and / or Z2 ) contains 5'-Z-VP.

[0714] In one embodiment, the single-stranded oligonucleotide comprises at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ) containing 5'-PS 2 .

[0715] In one embodiment, the single-stranded oligonucleotide comprises at least one nucleotide sequence (e.g., Z 1 and / or Z 2 ) containing 5'-deoxy-5'-C-malonyl.

[0716] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the single-stranded oligonucleotides are modified. For example, when 50% of the single-stranded oligonucleotides are modified, 50% of all the nucleotides present in the single-stranded oligonucleotides contain the modifications described herein.

[0717] In one embodiment, each nucleotide of Z 1 and Z 2 of the single-stranded oligonucleotide is independently modified by an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0718] In one embodiment, the nucleotide sequence (e.g., Z 1 and / or Z 2 ) of the single-stranded oligonucleotide contains at least two different modifications.

[0719] In one embodiment, the single-stranded oligonucleotide does not contain a 2'-F modification.

[0720] In one embodiment, the 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 a phosphorothioate nucleotide linkage or a methylphosphonate nucleotide linkage. For example, two blocks of phosphorothioate nucleotide linkages or methylphosphonate nucleotide linkages are separated by 16 to 18 phosphodiester nucleotide linkages.

[0721] In one embodiment, the nucleotide at the 1st position at the 5' end of the nucleotide sequence (e.g., Z 1 and / or Z 2 ) is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pairs from the 5' end of the nucleotide sequence (e.g., Z 1 and / or Z 2 ) is an AU base pair.

[0722] In one embodiment, the single-stranded oligonucleotide is 100% complementary to the target RNA, hybridizes thereto, 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.

[0723] In some embodiments herein, there is provided a single-stranded oligonucleotide capable of inhibiting the expression of a target gene. The single-stranded oligonucleotide contains at least one thermolabile nucleotide.

[0724] Thermally unstable nucleotides can occur, for example, between positions 14 and 17 at the 5' end of a nucleotide sequence of 21 nucleotides in length (e.g., Z 1 and / or Z 2 ). The nucleotide sequence can contain at least two modified nucleic acids that are smaller than the sterically required 2'-OMe modification. Preferably, the two modified nucleic acids that are smaller than the sterically required 2'-OMe are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are at positions 2 and 14 at the 5' end.

[0725] In some embodiments, the single-stranded oligonucleotide has the formula (II): 5’np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3’ (II) [wherein: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each Na independently represents an oligonucleotide sequence containing 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 represent overhanging nucleotides; wherein Nb and Y do not have the same modification; wherein XXX, YYY, and ZZZ each independently represent one motif by three identical modifications on three consecutive nucleotides] and contains a sequence represented by.

[0726] In some embodiments, the single-stranded oligonucleotide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications (plural available). In one example, the single-stranded oligonucleotide contains 9 or 10 2'-F modifications.

[0727] A single-stranded oligonucleotide may further comprise at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage. Modification by a phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage may occur at any nucleotide of the single-stranded oligonucleotide. For example, modification by an internucleotide linkage may occur at any nucleotide on at least one nucleotide sequence; modification by each internucleotide linkage may occur in an alternating pattern on at least one nucleotide sequence.

[0728] In some embodiments, the compounds of the invention disclosed herein are miRNA mimics. In one design, an miRNA mimic 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 having identity to the mature strand of a given miRNA. The double-stranded miRNA mimic has a design similar to that described above for double-stranded iRNAs. In some embodiments, the miRNA mimic comprises a double-stranded region of 16 to 31 nucleotides and one or more of the following chemical modification patterns: the sense strand has 2'-O-methyl modification of nucleotides 1 and 2 (counting from the 5' end of the sense strand), and contains all C's and U's, and the modification of the antisense strand may include 2'F modification of all C's and U's, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with a 2-nucleotide 3' overhang.

[0729] In some embodiments, the compounds of the invention disclosed herein are antimir. In some embodiments, the compounds of the invention comprise at least two antimirs covalently linked to each other or non-covalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linkers described in this disclosure. 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. The inhibitor can adopt various configurations including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplex), hairpin designs, and generally, a microRNA inhibitor comprises one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (or strands) of the targeted miRNA. Further, the miRNA inhibitor can also comprise additional sequences located 5′ and 3′ of a sequence that is the reverse complement of the mature miRNA. The additional sequences can be the reverse complement of the sequences adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequences can be any sequence (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences can be any sequence capable of forming a hairpin. Thus, in some embodiments, a sequence that is the reverse complement of the miRNA is flanked on the 5′ and 3′ sides by hairpin structures. A microRNA inhibitor can comprise mismatches between nucleotides on opposite strands in the case of a double-stranded molecule. Further, a microRNA inhibitor can be linked to a conjugate moiety to facilitate uptake of the inhibitor into cells.

[0730] MicroRNA inhibitors containing 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, each of which is incorporated herein by reference in its entirety. One of ordinary skill in the art can select a sequence from a database for a desired miRNA and design an inhibitor useful in the methods disclosed herein.

[0731] In some embodiments, the compounds of the invention disclosed herein are antagomirs. In some embodiments, the compounds of the invention comprise at least two antagomirs covalently linked to each other or non-covalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linkers described in this disclosure. An antagomir is an RNA-like oligonucleotide and has various modifications for RNAse protection and pharmacological properties, such as enhancing uptake into tissues and cells. They differ from normal RNA, for example, by complete 2'-O-methylation of sugars, phosphorothioate sugar-sugar linkages, and, for example, a cholesterol moiety at the 3' end. In a preferred embodiment, the antagomir comprises 2'-O-methyl modification at all nucleotides, a cholesterol moiety at the 3' end, two phosphorothioate sugar-strand linkages at the first two positions at the 5'-end, and four phosphorothioate linkages at the 3' end of the molecule. Antagomirs can be used to efficiently suppress endogenous miRNAs by forming a duplex containing the antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of miRNA silencing via an antagomir is the silencing of miR-122, which is described in Krutzfeldt et al, Nature, 2005, 438: 685-689 and is hereby expressly incorporated by reference in its entirety.

[0732] Recent studies have also found that dsRNA can activate gene expression, and this mechanism is called "small RNA-induced gene activation" or RNAa (activating RNA). See, for example, Li, L.C. et al. Proc Natl Acad Sci U S A. (2006), 103(46):17337-42, and Li L.C. (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. dsRNA targeting gene promoters has been shown to induce strong transcriptional activation of related genes. Endogenous miRNAs that cause RNAa have also been found in humans. Check E. Nature (2007). 448 (7156): 855-858.

[0733] Another surprising observation is that gene activation by RNAa persists for a long time. Induction of gene expression has been confirmed to last for more than 10 days. The long-lasting effect of RNAa may be due to epigenetic changes at the dsRNA target site. In some embodiments, the RNA activator can increase gene expression. In some embodiments, the increased gene expression inhibits viability, growth and development, and / or regeneration.

[0734] Accordingly, in some embodiments, the compounds of the invention disclosed herein are activating RNAs. In some embodiments, the compounds of the invention comprise at least two activating RNAs covalently linked to each other or non-covalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linkers described in this disclosure.

[0735] Accordingly, in some embodiments, the compounds of the invention disclosed herein are triplex-forming oligonucleotides (TFOs). In some embodiments, the compounds of the invention comprise at least two TFOs covalently linked to each other or non-covalently linked to each other via a nucleotide-based linker or a non-nucleotide-based linker, such as the linkers described in this disclosure. Recent studies have shown that triplex-forming oligonucleotides that sequence-specifically recognize and bind to polypurine / polypyrimidine regions in double-stranded helical DNA can be designed. These recognition rules are summarized in Maher III, L.J., et al., Science (1989) vol. 245, pp 725-730; Moser, H. E., et al., Science (1987) vol. 238, pp 645-630; Beal, P.A., et al., Science (1992) vol. 251, pp 1360-1363; Conney, M., et al., Science (1988) vol. 241, pp 456-459 and Hogan, M.E., et al., EP Publication 375408. Modifications of oligonucleotides such as the introduction of intercalators and substitution of sugar linkages, and optimization of binding conditions (pH and cation concentration) have helped to overcome the obstacles inherent in TFO activity such as charge repulsion and instability, and recently, it has been shown that synthetic oligonucleotides can be targeted to specific sequences (see the recent review by Seidman and Glazer, J Clin Invest 2003;l 12:487-94). Generally, triplex-forming oligonucleotides correspond to sequences such as: Oligo 3'-AGGT Duplex 5'-AGCT Duplex 3'-TCGA

[0736] However, it has been shown that the triplets A-AT and G-GC are the most stable in triple helices (Reither and Jeltsch, BMC Biochem, 2002, Sept 12, Epub). The same authors have shown that TFOs designed according to the rules of A-AT and G-GC do not form non-specific triple strands, indicating that triple-strand formation is actually sequence-specific.

[0737] Therefore, for any given sequence, a triple-strand-forming sequence can be devised. The triple-strand-forming oligonucleotide is preferably at least 15 nucleotides in length, more preferably 25 nucleotides in length, even more preferably 30 nucleotides or more in length, up to 50 or 100 nucleotides.

[0738] The formation of a triple-helix structure with the target DNA induces steric and functional changes, blocks the initiation and elongation of transcription, allows the introduction of desired sequence changes into the endogenous DNA, and results in specific downregulation of gene expression. Examples of such gene expression suppression in cells treated with TFOs include the knockout of episomal supFGl and the endogenous HPRT gene in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27: 1176-81, and Puri, et al, J Biol Chem, 2001;276:28991-98), the sequence-specific and target-specific downregulation of the Ets2 transcription factor, which is important in the etiology of prostate cancer (Carbone, et al, Nucl Acid Res. 2003 ;31:833-43), and the inflammation-inducible ICAM-I gene (Besch et al, J Biol Chem, 2002;277:32473-79). Furthermore, Vuyisich and Beal recently showed that sequence-specific TFOs bind to dsRNA and inhibit the activity of dsRNA-dependent enzymes such as RNA-dependent kinase (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

[0739] Furthermore, TFOs designed according to the above principles can induce targeted mutagenesis that can effectively perform DNA repair, and as a result, can both downregulate and upregulate the expression of endogenous genes (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 Application Nos. 2003 017068 and 2003 0096980 to Froehler et al., as well as U.S. Patent Application Nos. 2002 0128218 and 2002 0123476 to Emanuele et al., and U.S. Patent No. 5,721,138 to Lawn, the contents of which are hereby incorporated by reference in their entirety.

[0740] Nucleic acid modification In some embodiments, the single-stranded oligonucleotide comprises at least one nucleic acid modification described herein. For example, the at least one modification is selected from the group consisting of modified internucleoside linkages, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modifications can be present anywhere in the single-stranded oligonucleotide. For example, the modification can be present in one of the RNA molecules.

[0741] 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 containing a pentofuranosyl sugar, a phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In the formation of oligonucleotides, these phosphate groups covalently bond adjacent nucleosides to each other to form a linear polymeric compound. Within an oligonucleotide, the phosphate groups are generally said to form the internucleoside backbone of the oligonucleotide. The naturally occurring linkages or backbones of RNA and DNA are 3' to 5' phosphodiester linkages.

[0742] In addition to “unmodified” or “natural” nucleobases such as purine nucleobases adenine (A) and guanine (G), and pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for 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 with these bases, or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanosine, or tubercidin), and any one of the oligomer modifications described herein. Alternatively, substitution or modified analogs of any of the above bases and “universal bases” can be used. When natural bases are replaced by non-natural and / or universal bases, the nucleotides are said to contain modified nucleobases and / or the nucleobase modifications of this specification. Modified nucleobases and / or nucleobase modifications include natural, non-natural, and universal bases, and include conjugated moieties such as ligands described herein. Preferred conjugated moieties for conjugation to nucleobases include cationic amino groups that can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0743] The oligomeric compounds described herein may also include nucleobase (often simply referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nebularine, isoguanosine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6 -(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(Acetyl)Cytosine, 3-(3-Amino-3-carboxypropyl)Uracil, 2-(Thio)Uracil, 5-(Methyl)-2-(Thio)Uracil, 5-(Methylaminomethyl)-2-(Thio)Uracil, 4-(Thio)Uracil, 5-(Methyl)-4-(Thio)Uracil, 5-(Methylaminomethyl)-4-(Thio)Uracil, 5-(Methyl)-2,4-(Dithio)Uracil, 5-(Methylaminomethyl)-2,4-(Dithio)Uracil, 5-(2-Aminopropyl)Uracil, 5-(Alkyl)Uracil, 5-(Alkynyl)Uracil, 5-(Allylamino)Uracil, 5-(Aminoallyl)Uracil, 5-(Aminoalkyl)Uracil, 5-(Guanidiniumalkyl)Uracil, 5-(1,3-Diazol-1-alkyl)Uracil, 5-(Cyanoalkyl)Uracil, 5-(Dialkylaminoalkyl)Uracil, 5-(Dimethylaminoalkyl)Uracil, 5-(Halo)Uracil, 5-(Methoxy)Uracil, Uracil-5-oxyacetic Acid, 5-(Methoxycarbonylmethyl)-2-(Thio)Uracil, 5-(Methoxycarbonyl-methyl)Uracil, 5-(Propynyl)Uracil, 5-(Propynyl)Uracil, 5-(Trifluoromethyl)Uracil, 6-(Azo)Uracil, DihydroUracil, N 3-(Methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted-4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethenyl)-pseudouracil, 1-(aminocarbonylethenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-2,4-(dithio)pseudouracil, 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxo)-naphthalene, inosine, xanthine, hypoxanthine, nubralin, tubercidin, isoguanicin, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitrotriazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarboloxylilyl, 5-(methyl)isocarboloxylilyl, 3-(methyl)-7-(propynyl)isocarboloxylilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarboloxylilyl, 7-(propynyl)isocarboloxylilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)p...

Claims

1. Equation (I): (5’-Z １ -3’)-Q １ -L-Q ２ -(5’-Z ２ -3’) (I) [In the formula: Z １ This is a first oligonucleotide comprising 10 to 100 appropriately modified nucleotides that are substantially complementary to the target gene; Z ２ is, Z １ A second oligonucleotide comprising 10 to 100 appropriately modified nucleotides, substantially complementary to the first oligonucleotide; Z １ and Z ２ can form an intrastrand double-stranded region containing 3 or more consecutive base pairs; L is a linking group; Q １ and Q ２ Each of these independently represents 0 to 12 appropriately modified nucleotides. A single-stranded oligonucleotide by At least one nucleotide in formula (I) is a modified nucleotide. Single-stranded oligonucleotide.

2. L is the formula: #-(N) ｎ - ＊＊ [In the formula: # is Q １ It is a coupling to, ＊＊ Q ２ It is a coupling to; n is 3 to 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, comprising a linking portion represented by .

3. The single-stranded oligonucleotide according to claim 2, wherein one or more Ns are appropriately modified nucleotides.

4. The single-stranded oligonucleotide according to claim 3, wherein one or more Ns are independently selected from the group consisting of 2'-deoxynucleotide (dN), 2'-deoxy-2'-fluoronucleotide (fN), ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-aranucleotide (aN).

5. One or more N independently, 【Chemistry 1】 A single-stranded oligonucleotide according to claim 2, selected from the group consisting of the following.

6. One or more N independently, 【Chemistry 2】 [In the formula, Base is a nucleobase that has been modified as appropriate. R Ｄ C ４ ~ ３０ Alkyl, C ４ ~ ３０ Alkenyl, or C ４ ~ ３０ [It is Alkinnil] A single-stranded oligonucleotide according to claim 2, selected from the group consisting of the following.

7. The single-stranded oligonucleotide according to claim 2, comprising one or more Ns, optionally conjugated with a ligand, mono-, di-, tri-, tetra-, penta-, or polyprolinol; optionally conjugated with a ligand, mono-, di-, tri-, tetra-, penta-, or polyhydroxyprolinol; optionally modified nucleotide; or a combination thereof.

8. One or more N Table 1 A single-stranded oligonucleotide according to claim 7, comprising a portion selected from the group consisting of the following.

9. L is the formula: #-(N) ｎ - ＊＊ [In the formula: # is Q １ It is a coupling to, ＊＊ Q ２ It is a coupling to; n is between 3 and 10; and Each N is independently a 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. A single-stranded oligonucleotide according to claim 1, comprising a linking portion represented by .

10. The single-stranded oligonucleotide according to claim 9, wherein n is 4 to 8.

11. The single-stranded oligonucleotide according to claim 9, wherein n is 5.

12. The single-stranded oligonucleotide according to claim 9, wherein L contains three to five 2'-deoxynucleotides, a triplet of 2'-deoxy-2'-fluoronucleotides, a triplet of ribonucleotides, a triplet of 2'-O-methylnucleotides, or a triplet of Q304.

13. L, #-mN-mN-mN-mN-mN- ＊＊ 、 #-rN-rN-rN-rN-rN- ＊＊ 、 #-rN-rN-f ＊＊ 、 #-$N-$N-f ＊＊ 、 #-dN-rN-rN-rN-dN- ＊＊ 、 #-dN-dN-dN-dN-dN- ＊＊ 、 #-mN-mN-dN-dN-dN- ＊＊ 、 #-mN-mN-rN-dN-dN- ＊＊ 、 #-mN-mN-rN-rN-rN- ＊＊ , and #-mN-mN-f ＊＊ 、 [In the formula: 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 9, selected from the group consisting of the following.

14. L, #---mN-mN-Q304-Q304-Q304--- ＊＊ 、 #---dN-dN-Q304-Q304-Q304--- ＊＊ 、 #---dN-rN-Q304-Q304-Q304--- ＊＊ 、 #---rN-dN-Q304-Q304-Q304--- ＊＊ , and #---dN-rN-Q304-Q304-Q304--- ＊＊ 、 [In the formula: 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 9, selected from the group consisting of the following.

15. The single-stranded oligonucleotide according to claim 13 or 14, wherein one or more internucleotide links between nucleotides in L are independently 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).

16. The single-stranded oligonucleotide according to claim 1, further comprising one or more ligands.

17. The single-stranded oligonucleotide according to claim 1, wherein one or more of the five internucleotide links within the six 3'-terminal nucleotides or within the six 5'-terminal nucleotides are modified internucleotide links.

18. Z ２ The single-stranded oligonucleotide according to claim 1, wherein one or more of the five internucleotide links within the six 5'-terminal nucleotides of Z1 or within the six 5'-terminal nucleotides of Z1 are modified internucleotide links.

19. Z １ The 3'-terminal nucleotide and Q １ The single-stranded oligonucleotide according to claim 1, further comprising one or more modified nucleotide links between the first nucleotides and / or one or more modified nucleotide links between the nucleotides of Q1.

20. (a) Z １ and Z ２ Each of these independently contains 19 to 23 appropriately modified nucleotides; (b) Q １ and Q ２ Each 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 includes at least one modified nucleotide linkage; (h)Z １ It includes at least one modified nucleotide linkage; (i) The 5' terminal nucleotide contains a 5'-phosphate or 5'-phosphate mimetic modification; (j) The 3' terminal nucleotide is conjugated to a ligand via a linker as appropriate; (k)Z １ This means that it contains three or fewer mismatches with respect to the target gene; (l) L is the formula: #-(N) ｎ - ＊＊ [wherein n is 3 to 5, and each N independently contains a linkage represented by appropriately modified nucleotides Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, Q11, Q150, Q151, Q173, Q221, Q222, Q367, or Q368] A single-stranded oligonucleotide according to claim 1, characterized by one or more of the following.

21. (a) Z １ and Z ２ Each independently contains 21 appropriately modified nucleotides; (b) Q １ and Q ２ Each 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 ２ The 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 includes at least two sequentially modified nucleotide linkages; (h)Z １ It includes at least two sequentially modified nucleotide linkages; (i) Z １ The 5' terminal nucleotide contains a 5'-phosphate or 5'-phosphate mimetic modification; (j) Z ２ The 3' terminal nucleotide is conjugated to a ligand via a linker as appropriate; (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, and each N is independently a suitably modified nucleotide or Q304]. A single-stranded oligonucleotide according to claim 20, characterized by one or more of the following.

22. Z １ and Z ２ The single-stranded oligonucleotide according to claim 1, wherein each independently comprises 15 to 40 appropriately modified nucleotides.

23. Q １ and Q ２ The single-stranded oligonucleotide according to claim 1, wherein each of the nucleotides is independently 1 to 6 appropriately modified nucleotides.

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