Nucleic acid compound

Abstract

The present application provides novel nucleic acid compounds suitable for therapeutic use. In addition, the present application provides methods of making these compounds, and methods of using these compounds in the treatment of various diseases and conditions.

Description

Technical Field

[0001] The present application provides novel nucleic acid compounds suitable for therapeutic use. In addition, the present application provides methods for preparing these compounds, as well as methods for using these compounds to treat various diseases and conditions. Background of the Invention

[0003] Nucleic acid compounds have important therapeutic applications in medicine. Nucleic acids can be used to silence genes that cause specific diseases. Gene silencing prevents the formation of proteins by inhibiting translation. Importantly, gene silencers are promising alternatives to traditional small organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleotides / oligonucleosides that prevent protein formation through gene silencing.

[0004] Particularly in the past two decades, a large number of modified siRNA compounds for diagnostic and therapeutic purposes have been developed, including siRNA / RNAi therapeutics for treating various diseases, including central nervous system diseases, inflammatory diseases, metabolic disorders, oncology, infectious diseases, and ocular diseases.

[0005] The present application relates to nucleic acid compounds for use in treating and / or preventing diseases. Summary of the Invention

[0006] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand;

[0007] wherein the nucleosides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0008] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0009] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0010] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me.

[0011] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand;

[0012] The nucleosides of the second strand contain the following 2'-sugar and backbone modification patterns (5'-3'):

[0013] Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0014] Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0015] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0016] Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0017] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or

[0018] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0019] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0020] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0021] where (s) is a phosphorothioate internucleoside bond.

[0022] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand;

[0023] where the nucleosides of the second strand contain the following 2'-sugar and backbone modification patterns without bases (5'-3'):

[0024] ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0025] ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0026] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0027] ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0028] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, or

[0029] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0030] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0031] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0032] wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia–ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0033] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand; wherein the nucleosides of the second strand comprise the following 2'-sugar, abasic, and linkage modification patterns (5'-3'):

[0034] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0035] ia–ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0036] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0037] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0038] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia, or

[0039] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or

[0040] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or

[0041] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0042] Wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0043] The nucleic acids described herein generally comprise a first strand that contains a modification pattern selected from the following or any combination thereof, where position 1 is the 5'-terminal nucleoside of the first strand and the counting direction is 5'–3':

[0044] 2'-F sugar modification at least at positions 2, 14, and 16, and / or

[0045] a 2'-Me sugar modification at positions 17 to 23, or the first strand comprises at least eight 2'-F sugar modifications, such as 2'-F sugar modifications at least at positions 2, 4, 6, 12, 14, 16, 18, and 20, and / or

[0046] a 2'-Me sugar modification at positions 1, 3 to 5, 10 to 13, or the first strand comprises at least eight 2'-F sugar modifications, such as 2'-F sugar modifications at least at positions 2, 4, 6, 12, 14, 16, 18, and 20, and / or

[0047] a 2'-Me sugar modification or a thermally destabilizing modification at position 7, such as a locked nucleic acid or glycol nucleic acid modified typically at position 7, and / or

[0048] a 2'-F sugar modification or a thermally destabilizing modification at position 6, such as a locked nucleic acid or glycol nucleic acid modified typically, and / or

[0049] Positions 8 and 9 can be modifications selected from 2'-Me sugar modifications and 2'-F sugar modifications, and can typically be the same 2'-sugar modification,

[0050] Thus, typically, the first strand can comprise the following modification pattern (5'-3'):

[0051] Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0052] where M represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and thermally destabilizing modification (such as a locked nucleic acid or glycol nucleic acid modified typically), which is typically present at position 6, and typically a 2'-Me sugar modification is present at position 7,

[0053] Or typically, the first strand comprises the following modification pattern (5'-3'):

[0054] Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0055] where M1 represents a thermally destabilizing modification, such as a locked nucleic acid or glycol nucleic acid modified typically, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification,

[0056] Or typically, the first strand comprises the following modification pattern (5'-3'):

[0057] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me

[0058] wherein M1 represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat destabilizing modification (such as generally modified unlocked nucleic acid or ethylene glycol nucleic acid), and M2 represents a modification selected from the following: 2'-Me sugar modification and 2'-F sugar modification,

[0059] or generally the first strand comprises the following modification pattern (5'-3'):

[0060] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0061] wherein M1 represents a heat destabilizing modification, such as generally modified unlocked nucleic acid or ethylene glycol nucleic acid, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification, and generally M2 can be the same 2' sugar modification.

[0062] Generally, (M)4 as described above represents any of the following 2' sugar modification patterns (5'–3'):

[0063] F–Me–Me–F

[0064] Me–F–Me–F

[0065] F–Me–F–Me

[0066] F–F–F–F

[0067] Me–F–F–Me

[0068] Me–Me–F–F

[0069] F–F–Me–Me

[0070] Me–Me–Me–Me

[0071] Typically, as described herein, two internucleoside phosphorothioate linkages are present between three consecutive positions in the 5' and 3' terminal regions of the first strand, whereby the terminal nucleosides in each of the 5' and 3' terminal regions of the first strand are each linked via an internucleoside phosphorothioate linkage to their respective 5' and 3' adjacent penultimate nucleosides, and each 5' and 3' penultimate nucleoside is linked via an internucleoside phosphorothioate linkage to their respective 5' and 3' adjacent antepenultimate nucleosides, and if appropriate, two internucleoside phosphorothioate linkages may also be present between three consecutive positions in the 3' terminal region of the second strand, whereby the 3' terminal nucleoside is linked via an internucleoside phosphorothioate linkage to the adjacent penultimate nucleoside, and the penultimate nucleoside is linked via an internucleoside phosphorothioate linkage to the adjacent antepenultimate nucleoside, and / or if appropriate, two internucleoside phosphorothioate linkages may also be present between three consecutive positions in the 5' terminal region of the second strand, whereby the 5' terminal nucleoside is linked via an internucleoside phosphorothioate linkage to the adjacent penultimate nucleoside, and the penultimate nucleoside is linked via an internucleoside phosphorothioate linkage to the adjacent antepenultimate nucleoside.

[0072] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the nucleosides of the second strand comprise the following 2'-sugar and abasic modification pattern (5'-3'):

[0073] Second strand (5'-3'): ia-ia-Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me

[0074] Or position 7 on the second strand comprises a sugar modification (i.e., 2'-Me modification), wherein position 1 is the 5' terminal nucleoside of the second strand, the counting direction is 5'–3', and generally two inverted abasic nucleosides are present in the 5' terminal region of the second strand,

[0075] wherein such a second strand is generally used in combination with the first strand as defined herein.

[0076] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0077] Modification pattern 1:

[0078] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me,

[0079] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0080] Or modification pattern 2:

[0081] Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me

[0082] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0083] Or modification pattern 3:

[0084] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0085] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0086] Or modification pattern 4:

[0087] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0088] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0089] Or modification pattern 5:

[0090] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0091] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0092] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar modification patterns (5’-3’):

[0093] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0094] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0095] or

[0096] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0097] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0098] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and bond modification patterns (5’-3’):

[0099] (5’-3’): Modification pattern 1:

[0100] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me,

[0101] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0102] Or modification pattern 2:

[0103] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0104] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0105] Or modification pattern 3:

[0106] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0107] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0108] Or modification pattern 4:

[0109] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0110] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0111] Or modification pattern 5:

[0112] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0113] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0114] or modification pattern 6:

[0115] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0116] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0117] where (s) is a phosphorothioate internucleoside linkage.

[0118] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and linkage modification patterns (5’-3’):

[0119] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0120] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0121] or

[0122] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0123] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0124] where (s) is a phosphorothioate internucleoside linkage.

[0125] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region that includes: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and linkage modification patterns (5'-3'):

[0126] (5'-3'): Modification pattern 1:

[0127] Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me,

[0128] First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0129] Or modification pattern 2:

[0130] Second strand (5'-3'): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0131] First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0132] Or modification pattern 3:

[0133] Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0134] First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0135] Or modification pattern 4:

[0136] Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0137] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0138] or modification pattern 5:

[0139] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0140] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0141] or modification pattern 6:

[0142] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0143] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0144] where (s) is a phosphorothioate internucleoside bond.

[0145] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and base-free modification patterns (5’-3’):

[0146] Modification pattern 1:

[0147] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me,

[0148] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0149] Or modification pattern 2:

[0150] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0151] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0152] Or modification pattern 3:

[0153] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0154] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0155] Or modification pattern 4:

[0156] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0157] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0158] Or modification pattern 5:

[0159] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0160] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0161] Or modification pattern 6:

[0162] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me–Me

[0163] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0164] Wherein ia represents an inverted abasic nucleoside.

[0165] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and abasic modification patterns (5’-3’):

[0166] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0167] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0168] or

[0169] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0170] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0171] Wherein ia represents an inverted abasic nucleoside.

[0172] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and abasic modification patterns (5’-3’):

[0173] Modification mode 1:

[0174] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia,

[0175] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0176] Or modification mode 2:

[0177] Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0178] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0179] Or modification mode 3:

[0180] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0181] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0182] Or modification mode 4:

[0183] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0184] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0185] Or modification mode 5:

[0186] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0187] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0188] Or modification pattern 6:

[0189] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia

[0190] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me,

[0191] Wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0192] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2’ sugar, abasic, and linkage modification patterns (5’-3’):

[0193] Modification pattern 1:

[0194] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me,

[0195] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0196] Or modification pattern 2:

[0197] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[0198] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0199] Or modification pattern 3:

[0200] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[0201] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0202] Or modification pattern 4:

[0203] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0204] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0205] Or modification pattern 5:

[0206] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0207] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0208] Or modification pattern 6:

[0209] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[0210] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0211] wherein: (s) is a phosphorothioate internucleoside linkage, and ia represents an inverted abasic nucleoside.

[0212] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2’ sugar, abasic, and linkage modification patterns (5’-3’):

[0213] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0214] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0215] or

[0216] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0217] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0218] wherein: (s) is a phosphorothioate internucleoside linkage, and ia represents an inverted abasic nucleoside.

[0219] A nucleic acid for inhibiting the expression of ZPI or HCII, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from ZPI or HCII, and a second strand at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar, abasic and bond modification patterns (5'-3'):

[0220] Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0221] First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0222] wherein: (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside.

[0223] A nucleic acid for inhibiting the expression of B4GALT1, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from B4GALT1, and a second strand at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar, abasic and bond modification patterns (5'-3'):

[0224] Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0225] First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0226] wherein: (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside.

[0227] A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar, abasic and bond modification patterns (5'-3'):

[0228] Modification pattern 1:

[0229] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia,

[0230] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0231] Or modification pattern 2:

[0232] Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0233] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0234] Or modification pattern 3:

[0235] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0236] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0237] Or modification pattern 4:

[0238] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0239] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0240] Or modification pattern 5:

[0241] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0242] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0243] Or modification pattern 6:

[0244] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0245] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0246] Wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia–ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0247] Particularly suitable nucleic acids for use in the present application for inhibiting the expression of a target gene, which comprise a duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand;

[0248] Wherein, the second strand comprises, starting from position 1 at the 5’ end of the second strand (excluding the nucleoside closest to the 5’ of the abasic nucleoside), position 7 on the second strand comprises a sugar modification (the sugar modification is a 2’-Me modification), or the second strand comprises the following modification pattern:

[0249] Second strand (5’-3’): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me

[0250] Particularly suitable nucleic acids in the present application are as follows:

[0251] Second strand (5’-3’): ia–ia-Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me

[0252] which, together with a first strand having a modification pattern selected from the following (5’-3’), wherein position 1 is the 5’-terminal nucleoside of the first strand and the counting direction is (5’-3’):

[0253] (5’-3’)Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0254] wherein M represents a modification selected from: a 2’-Me sugar modification, a 2’-F sugar modification, and a heat destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid, typically), which is usually present at position 6, and a 2’-Me sugar modification is usually present at position 7,

[0255] or alternatively the first strand typically comprises the following modification pattern (5’-3’):

[0256] (5’-3’)Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0257] wherein M1 represents a heat destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid, typically, and M2 represents a modification selected from a 2’-Me sugar modification and a 2’-F sugar modification,

[0258] or alternatively the first strand typically comprises the following modification pattern (5’-3’):

[0259] (5’-3’)Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0260] wherein M1 represents a modification selected from: a 2’-Me sugar modification, a 2’-F sugar modification, and a heat destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid, typically), and M2 represents a modification selected from a 2’-Me sugar modification and a 2’-F sugar modification,

[0261] or alternatively the first strand typically comprises the following modification pattern (5’-3’):

[0262] (5’-3’)Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0263] Wherein M1 represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid, M2 represents a modification selected from 2’-Me sugar modification and 2’-F sugar modification, and generally M2 can be the same 2’ sugar modification.

[0264] A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the second strand comprises: 2 consecutive abasic nucleosides within the 5’ or 3’ terminal region of the second strand, and at the 7th position on the second strand counting from the 5’ terminal position 1 (excluding the nucleoside closest to the 5’ of the abasic nucleoside), there is a sugar modification, which is a 2’-Me modification.

[0265] Particularly suitable according to the present application is a nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the second strand and the first strand comprise the following modification patterns:

[0266] Second strand (5’-3’): ia–ia-Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me

[0267] First strand (5’-3’): Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0268] Wherein M1 represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid, M2 represents a modification selected from the following: 2’-Me sugar modification and 2’-F sugar modification, and generally M2 can be the same 2’ sugar modification.

[0269] A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand comprises: two phosphorothioate internucleoside bonds respectively present between positions 1 and 2 and between 2 and 3 of the second strand, counted from the 5'-terminal position 1 of the second strand (excluding the nucleoside closest to the 5'-end that is a baseless nucleoside), or two phosphorothioate internucleoside bonds respectively present between positions 1 and 2 and between 2 and 3 of the second strand, counted from the 3'-terminal position 1 of the second strand (excluding the nucleoside closest to the 3'-end that is a baseless nucleoside), and at position 7 on the second strand, counted from the 5'-terminal position 1 of the second strand (excluding the nucleoside closest to the 5'-end that is a baseless nucleoside), there is a sugar modification, which is a 2'-Me modification.

[0270] A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand comprises: at position 7 on the second strand, counted from the 5'-terminal position 1 (which is the nucleoside closest to the 5'-end excluding the baseless nucleoside), there is a sugar modification (the sugar modification is a 2'-Me modification), and at the 3'-terminal of the second strand, the nucleic acid is directly or indirectly conjugated to one or more ligand moieties, wherein the ligand moiety preferably comprises: one or more N-acetylgalactosamine (GalNAc) ligands, and / or one or more derivatives of N-acetylgalactosamine (GalNAc) ligands, and / or one or more N-acetylgalactosamine (GalNAc) ligands and / or their derivatives, which are conjugated to the nucleic acid through a linker.

[0271] Each of the above second strand sequences and constructs can be used together with any of the first strands described herein.

[0272] A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand has the following modification pattern:

[0273] Modification pattern 1:

[0274] Second strand (5'-3'): ia–ia–F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F, optionally in combination with the following first strand:

[0275] First strand (5’-3’): Me–F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F–Me,

[0276] or modification pattern 2:

[0277] Second strand (5’-3’): F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F-ia–ia, optionally combined with the following first strand:

[0278] First strand (5’-3’): Me–F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F–Me

[0279] wherein: ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0280] A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand has the following modification patterns:

[0281] Modification pattern 1:

[0282] Second strand (5’-3’): ia–ia–F(s)Me(s)F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F, optionally combined with the following first strand:

[0283] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me(s)F(s)Me,

[0284] or modification pattern 2:

[0285] Second strand (5’-3’): F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me–F(s)Me(s)F-ia–ia, optionally combined with the following first strand:

[0286] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me(s)F(s)Me

[0287] Wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia–ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0288] Other nucleic acids according to the present application include nucleic acids for inhibiting the expression of a target gene, which comprise a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the nucleosides of the second strand and the first strand comprise the following 2’ sugar and bond modification patterns (5’-3’):

[0289] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0290] Wherein the 2’-Me or 2’-F modified nucleosides of the first strand comprise any of the following modification patterns (5’-3’):

[0291] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0292] First strand (5’-3’): Me–F–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0293] First strand (5’-3’): Me–F–Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0294] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0295] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0296] First strand (5’-3’): Me–F–Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0297] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me,

[0298] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0299] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0300] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0301] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0302] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0303] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0304] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me,

[0305] wherein: (s) is a phosphorothioate internucleoside bond.

[0306] Other nucleic acids according to the present application include nucleic acids for inhibiting the expression of a target gene, which comprise a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar, abasic, and linkage modification patterns (5’-3’):

[0307] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0308] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0309] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0310] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0311] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0312] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia

[0313] wherein, the 2’-Me or 2’-F modified nucleosides of the first strand include any one of the following modification patterns (5’-3’):

[0314] First strand (5’-3’): Me–F–Me–Me–Me–F–Me–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0315] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0316] First strand (5’-3’): Me–F–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0317] First strand (5’-3’): Me–F–Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0318] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0319] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0320] First strand (5’-3’): Me–F–Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[0321] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me,

[0322] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–Me–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0323] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0324] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0325] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0326] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0327] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0328] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[0329] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me,

[0330] Wherein:

[0331] (s) is a phosphorothioate internucleoside bond,

[0332] ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0333] The nucleic acid according to the present application may further comprise a first strand, the first strand comprising at least 17 consecutive nucleosides, the consecutive nucleosides differing from any of the first strand sequences listed in Table 2 by 0 or 1 nucleoside.

[0334] The nucleic acid according to the present application may also comprise a first strand, which comprises at least 17 consecutive nucleosides that differ from any of the first strand sequences listed in Table 3 by 0 or 1 nucleoside.

[0335] Generally, the first strand as described above comprises nucleosides 2-18 of any of the sequences defined in Table 2 or Table 3.

[0336] The nucleic acid according to the present application may also comprise a second strand, which comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any of the second strand sequences listed in Table 2 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleosides.

[0337] The nucleic acid according to the present application may also comprise a second strand, which comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any of the second strand sequences listed in Table 2 by 0 or 1 nucleoside, and wherein the duplex region comprises at least 14, 15, 16 or 17 complementary base pairs.

[0338] The nucleic acid according to the present application may also comprise a second strand, which comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any of the second strand sequences listed in Table 4 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleosides.

[0339] The nucleic acid according to the present application may also comprise a second strand, which comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any of the second strand sequences listed in Table 4 by 0 or 1 nucleoside, and wherein the duplex region comprises at least 14, 15, 16 or 17 complementary base pairs.

[0340] The nucleic acid according to the present application, wherein the first strand comprises any of the first strand sequences listed in Table 2.

[0341] The nucleic acid according to the present application, wherein the first strand comprises any of the first strand sequences listed in Table 3.

[0342] The nucleic acid according to the present application, wherein the second strand comprises any of the second strand sequences listed in Table 2.

[0343] The nucleic acid according to the present application, wherein the second strand comprises any of the second strand sequences listed in Table 4.

[0344] The nucleic acid according to the present application, wherein the first strand and the second strand form any of the duplexes listed in Table 5.

[0345] The nucleic acid according to the present application, wherein the nucleic acid is an siRNA oligonucleoside.

[0346] A nucleic acid according to the present application, wherein the nucleic acid is directly or indirectly coupled to one or more ligand moieties, optionally, wherein the ligand moiety is present in the terminal region of the second strand, typically present in its 3'-terminal region, and typically may comprise one or more N-acetylgalactosamine (GalNAc) ligands, and / or one or more derivatives of N-acetylgalactosamine (GalNAc) ligands, and / or one or more N-acetylgalactosamine (GalNAc) ligands and / or their derivatives, which are coupled to the nucleic acid via a linker. Typically, one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly coupled to the 5'- or 3'-terminal region of the second strand of the nucleic acid, typically coupled to its 3'-terminal region.

[0347] A nucleic acid according to the present application, comprising a ligand moiety having the following structure:

[0348]

[0349] A nucleic acid according to the present application, comprising a ligand moiety having the following structure:

[0350]

[0351] wherein:

[0352] R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl each time it appears;

[0353] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro;

[0354] X1 and X2 are independently selected from the group consisting of methylene, oxygen, and sulfur each time they appear;

[0355] m is an integer from 1 to 6;

[0356] n is an integer from 1 to 10;

[0357] q, r, s, t, v are independently integers from 0 to 4, provided that: q and r cannot be 0 simultaneously; and s, t, and v cannot be 0 simultaneously;

[0358] Z is an oligonucleoside.

[0359] A nucleic acid according to the present application, comprising the structure:

[0360]

[0361] wherein [oligonucleotide] represents consecutive nucleosides of the second strand.

[0362] Alternatively, the nucleic acid according to the present application comprises a ligand moiety having the following structure:

[0363]

[0364] wherein: r and s are independently integers from 1 to 16; and

[0365] Z is an oligonucleoside.

[0366] The nucleic acid according to the present application comprises the structure:

[0367]

[0368] wherein [oligonucleotide] represents consecutive nucleosides of the second strand.

[0369] The present application also provides a pharmaceutical composition comprising the nucleic acid described herein and a pharmaceutically acceptable excipient or carrier.

[0370] The present application also provides the use of the nucleic acid or pharmaceutical composition described herein for treatment.

[0371] The present application also provides the use of the nucleic acid or pharmaceutical composition described herein for the prevention or treatment of a disease associated with a hemostatic disorder, such as hemophilia.

[0372] The present application also provides the use of the nucleic acid or pharmaceutical composition described herein for the prevention or treatment of cardiovascular diseases.

[0373] The present application also provides the use of the nucleic acid or pharmaceutical composition described herein for the prevention or treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0374] Figure 1 : Linkers and ligand moieties suitable for constructs of the present application, including tether 1a. Although Figure 1 linkers conjugated to oligonucleotides are described, it should be understood that the present application also encompasses conjugates of the same linkers with the oligonucleosides disclosed herein.

[0375] It should also be understood that although Figure 1 products of linkers and ligand moieties conjugated to oligonucleoside moieties as specifically described in Figure 1 are described, the product may alternatively comprise or consist essentially of a molecule in which the linker and ligand moieties are conjugated to the oligonucleoside moiety substantially as Figure 1 shown, but having an F substituent on the cyclooctyl ring as shown in Figure 1 and replaced by a substituent (such as an OH substituent) resulting from hydrolysis of the F substituent. In this way, (a) the tether 1a construct may consist essentially of having specifically as Figure 1The molecular composition of the linker and ligand moieties shown, where the cyclooctyl ring has an F substituent; or (b) the tether 1a construct may consist essentially of molecules having the linker and ligand moieties as specifically described in Figure 1 , but where Figure 1 the F substituent on the cyclooctyl ring shown is replaced by a substituent (e.g., an OH substituent) resulting from hydrolysis displacement, or (c) the tether 1a construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0376] Figure 2 : The linker and ligand moieties of constructs applicable to this application, including tether 1b. Although Figure 2 linkers conjugated to oligonucleotides are described, it should be understood that this application also encompasses conjugates of the same linkers with the oligonucleosides disclosed herein.

[0377] Regarding Figure 1 the description made and Figure 1 the possible replacements where the F substituent on the cyclooctyl ring shown is replaced by a substituent (e.g., an OH substituent) resulting from hydrolysis displacement are equally applicable to the tether 1b construct. In this way, (a) the tether 1b construct may consist essentially of molecules having the linker and ligand moieties as specifically described in Figure 2 , where the cyclooctyl ring has an F substituent; or (b) the tether 1b construct may consist essentially of molecules having the linker and ligand moieties as specifically described in Figure 2 , but Figure 2 the F substituent on the cyclooctyl ring described is replaced by a substituent (e.g., an OH substituent) resulting from hydrolysis displacement, or (c) the tether 1b construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0378] Figure 3 : The linker and ligand moieties of constructs applicable to this application, including tether 2a. Although Figure 3 linkers conjugated to oligonucleotides are described, it should be understood that this application also encompasses conjugates of the same linkers with the oligonucleosides disclosed herein.

[0379] Figure 4 : The linker and ligand moieties of constructs applicable to this application, including tether 2b. Although Figure 4 linkers conjugated to oligonucleotides are described, it should be understood that this application also encompasses conjugates of the same linkers with the oligonucleosides disclosed herein.

[0380] Figure 5 : The formulas described in items 1 - 101 disclosed herein.

[0381] Figure 6 : The formulas described in clauses 1 - 56 disclosed herein.

[0382] Figure 7a and 7b : Inverted abasic constructs that can be used with the nucleic acid sequences of the present application described herein. For Figure 7a , the GalNAc linker is attached to the 5'-end region of the sense strand used ( Figure 7a , not described in Figure 7b ). For Figure 7b , the GalNAc linker is attached to the 3'-end region of the sense strand used (

[0383] Figure 7a , not described in

[0384] Figure 7b ). The "iaia" shown in the 3'-end region of the sense strand in

[0385] means: (i) two abasic nucleosides as the penultimate and terminal nucleosides of the 3'-end region of the sense strand; (ii) a 3'-3' reverse linkage between the third-to-last nucleoside of the sense strand (i.e., at position 21 of the sense strand, where position 1 is the terminal 5'-nucleoside of the sense strand) and the adjacent penultimate abasic residue; and (iii) a 5'-3' bond between the terminal and penultimate abasic nucleosides when read towards the 3'-end region containing the terminal and penultimate abasic nucleosides.

[0386] Figure 8 (8a and 8b): Duplex constructs according to Table 5.

[0387] Figure 10 : Inhibition of ZPI expression by ETXM1184 (ETXS1036 & ETXS1035) and ETXM1199 (ETXS2398 & ETXS2397).

[0388] Figure 11 : Inhibition of B4GALT1 expression by ETXM1200 (ETXS2400 & ETXS2399) and ETXM1201 (ETXS2402 & ETXS2401).

[0389] Figure 12 : Inhibition of B4GALT1 expression by ETXM1203 (ETXS2406 & ETXS2405) and ETXM1204 (ETXS2408 & ETXS2407).

[0390] Figure 13 : Joint protection: Several endpoints recorded dose - response effects. Prophylactic administration of ETXM1184 showed dose - dependent protection in key tissue readings 10 days after injury. ETXM1184 showed the same range of efficacy as clinical controls: FVIII replacement therapy (the gold standard for emergency treatment (Benefix)) and an siRNA - based prophylactic rebalancing agent (fitusiran) showed good bleeding protection in late - stage clinical development. * Grading: 0 = normal; 1 = mild; 2 = moderate; 3 = marked; 4 = severe. [1] Glasson et al., Osteoarthritis Cartilage. 2010 Oct; 18 Suppl 3:S17 - 23.doi:10.1016 / j.joca.2010.05.025.PMID:20864019.

[0391] Figure 14 : Quantification of the comprehensive joint hematoma histopathology score: Tendinitis, tendon degeneration, tenosynovitis, periostitis, osteolysis, osteoclastic bone resorption, hemorrhage in vivo, hematoma, hemosiderosis, chondrocyte necrosis, cartilage OARSI grade, subchondral bone sclerosis, and myeloproliferation. ETX - 148 showed a significant dose - response effect (Bayesian linear model - fitted comprehensive score). Compared with the control group, the median reduction in the comprehensive score was: 1.25 in the ETXM1184 10 mg / kg group (significance level equivalent to p < 0.01); 0.91 in the ETXM1184 3 mg / kg group (significance level equivalent to p < 0.05). The control fitusiran showed a median reduction of 1.04 in the 3 mg / kg group (significance level equivalent to p < 0.05).

[0392] Figure 15 : Prophylactic administration of ETXM1184 improved the joint hematoma morbidity in hemophilia A mice. Administration of 3 mg / kg ETXM1184 improved knee joint hematoma morbidity, reduced inflammation, and reduced the bleeding area.

[0393] Figure 16 : Prophylactic administration of ETXM1184 reduces bleeding after injury in hemophilia A mice (in-life visual bleeding score (VBS)). Eight days after siRNA administration, a bleeding event occurred in the knee joints of hemophilia A mice. Bleeding was monitored within 10 days after injury and terminal histological analysis was performed. Prophylactic administration of a single dose of 10 mg / kg ETXM1184 effectively reduced the visual bleeding score (VBS) within 10 days after injury, which was comparable to that of factor VIII replacement (Benefix).

[0394] Figure 17 : Prophylactic administration of ETXM1184 reduces bleeding after knee joint injury in hemophilia A mice (in-life measurement of the diameter of the injured knee compared to the uninjured knee). Eight days after siRNA administration, a bleeding event occurred in the knee joints of hemophilia A mice. Bleeding was monitored within 10 days after injury and terminal histological analysis was performed. Prophylactic administration of a single dose of 10 mg / kg ETXM1184 effectively reduced blood accumulation in the knee joint within 10 days after injury, which was comparable to that of factor VIII replacement (Benefix).

[0395] Figure 18 : Prophylactic administration of ETXM1184 reduces joint hematoma in a hemophilia A mouse model (terminal measurements were taken 18 days after siRNA administration and 10 days after injury). Prophylactic administration of a single dose of 10 mg / kg ETXM1184 effectively reduced joint bleeding and the characteristics of hemophilic arthropathy within 10 days after injury, which was comparable to that of factor VIII replacement (Benefix). Detailed Description

[0396] Definitions

[0397] The "first strand" is also referred to herein as the antisense strand or guide strand, which are used interchangeably herein, and refers to a nucleic acid strand, such as a strand of siRNA (e.g., dsiRNA), which includes a region that is substantially complementary to a target sequence (e.g., mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence). When the complementary region is not completely complementary to the target sequence, the mismatches can generally be in the internal or terminal regions of the molecule. In some embodiments, the double-stranded nucleic acid (e.g., the siRNA agent of the present application) includes nucleoside mismatches in the antisense strand.

[0398] The "second strand" (also referred to herein as the sense strand or passenger strand, which can be used interchangeably herein) refers to a strand of nucleic acid, such as siRNA, which includes a region that is substantially complementary to the region of the antisense strand as defined herein.

[0399] In the context of a molecule comprising a nucleic acid having a ligand moiety and optionally also a linker, the nucleic acids of the present application may be referred to as oligonucleosides or oligonucleoside moieties.

[0400] Oligonucleotides are short nucleic acid polymers. Although oligonucleotides contain phosphodiester bonds between their nucleoside components (base plus sugar), the present application is not limited to oligonucleotides always linked by such phosphodiester bonds between adjacent nucleosides, and other nucleoside oligomers linked by bonds other than phosphodiester bonds are also contemplated. For example, the bond between nucleosides may be a phosphorothioate bond. Thus, the term "oligonucleoside" as used herein encompasses oligonucleotides and other nucleoside oligomers. According to the present application, oligonucleosides are preferably nucleic acids having at least a part that is an oligonucleotide. According to the present application, oligonucleosides having one or more or most of the phosphodiester backbone bonds between nucleosides are also preferred. According to the present application, oligonucleosides having one or more or most of the phosphodiester backbone bonds between nucleosides and also having one or more phosphorothioate backbone bonds between nucleosides (usually in the terminal regions of the first and / or second strands) are also preferred.

[0401] Preferably herein, the nucleic acids according to the present application are double-stranded oligonucleosides that contain one or more phosphorothioate backbone bonds between nucleosides. Thus, in all cases where oligonucleotides are mentioned in the present application, particularly in the chemical structures disclosed herein, the oligonucleotides may equally be oligonucleosides as defined herein.

[0402] In some embodiments, the double-stranded nucleic acids (such as siRNA agents) of the present application include nucleoside mismatches in the sense strand. In some embodiments, the nucleoside mismatches are, for example, within 5, 4, 3, 2, or 1 nucleoside from the 3' end of the nucleic acid (such as siRNA).

[0403] In another embodiment, the nucleoside mismatch is, for example, in the 3' terminal nucleoside of the nucleic acid (such as siRNA).

[0404] A "target sequence" (which may also be referred to as target RNA or target mRNA) refers to a continuous portion of the nucleoside sequence of an mRNA molecule formed during gene transcription, including mRNA, which is a product of the primary transcript of RNA processing.

[0405] The length of the target sequence can be about 10 - 35 nucleotides, such as about 15 - 30 nucleotides. For example, the length of the target sequence can be about 15 - 30 nucleotides, 15 - 29, 15 - 28, 15 - 27, 15 - 26, 15 - 25, 15 - 24, 15 - 23, 15 - 22, 15 - 21, 15 - 20, 15 - 19, 15 - 18, 15 - 17, 18 - 30, 18 - 29, 18 - 28, 18 - 27, 18 - 26, 18 - 25, 18 - 24, 18 - 23, 18 - 22, 18 - 21, 18 - 20, 19 - 30, 19 - 29, 19 - 28, 19 - 27, 19 - 26, 19 - 25, 19 - 24, 19 - 23, 19 - 22, 19 - 21, 19 - 20, 20 - 30, 20 - 29, 20 - 28, 20 - 27, 20 - 26, 20 - 25, 20 - 24, 20 - 23, 20 - 22, 20 - 21, 21 - 30, 21 - 29, 21 - 28, 21 - 27, 21 - 26, 21 - 25, 21 - 24, 21 - 23, or 21 - 22 nucleotides. Ranges and lengths between the above ranges and lengths are also considered part of the present application.

[0406] The term "ribonucleoside" or "nucleoside" may also refer to modified nucleosides, as further detailed below.

[0407] The nucleic acid can be DNA or RNA and can contain modified nucleosides. RNA is the preferred nucleic acid.

[0408] The terms "iRNA", "siRNA", "RNAi agent" and "iRNA agent", "RNA interference agent", which are used interchangeably herein, refer to an RNA-containing agent that mediates the targeted cleavage of an RNA transcript through the RNA-induced silencing complex (RISC) pathway. siRNA directs the sequence-specific degradation of mRNA through RNA interference (RNAi).

[0409] Double-stranded RNA is referred to herein as "double-stranded siRNA (dsiRNA) agent", "double-stranded siRNA (dsiRNA) molecule", "double-stranded RNA (dsRNA) agent", "double-stranded RNA (dsRNA) molecule", "dsiRNA agent", "dsiRNA molecule" or "dsiRNA", which refers to a complex of ribonucleic acid molecules having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands and having "sense" and "antisense" orientations relative to the target RNA.

[0410] Most of the nucleosides of each strand of the nucleic acid (e.g., dsiRNA molecule) are preferably ribonucleosides, but in such cases, one or more non-ribonucleosides, such as deoxyribonucleosides or modified nucleosides, may also be included in each strand or both strands. In addition, as used in this specification, "siRNA" may include ribonucleosides with chemical modifications.

[0411] The term "modified nucleoside" refers to a nucleoside independently having a modified sugar moiety, a modified internucleoside bond, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleoside" encompasses substitution, addition, or removal (e.g.) of functional groups or atoms to the internucleoside bond, sugar moiety, or nucleobase. For the purposes of this specification and claims, any such modification (as used in siRNA-type molecules) is encompassed within "iRNA" or "RNAi agent" or "siRNA" or "siRNA agent".

[0412] The two strands forming the duplex structure can be different parts of a larger molecule, or they can be separate molecules, such as RNA molecules.

[0413] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the duplex structure of the nucleic acid of the present application. The nucleic acid according to the present application may include an overhang of at least one nucleoside; alternatively, the overhang may include at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more. The nucleoside overhang may include or consist of nucleoside / nucleoside analogs (including deoxynucleosides). The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleosides of the overhang may be present at the 5'-end, 3'-end, or both ends of the antisense strand or the sense strand.

[0414] In certain embodiments, the antisense strand has 1 - 10 nucleosides, such as 0 - 3, 1 - 3, 2 - 4, 2 - 5, 4 - 10, 5 - 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, and the overhang is at the 3'-end or 5'-end.

[0415] "Blunt" or "blunt end" means that the end of the double-stranded nucleic acid has no unpaired nucleosides, i.e., no nucleoside overhang. The nucleic acids of the present application include nucleic acids having no nucleoside overhang at one end or no nucleoside overhang at each end.

[0416] Unless otherwise specified, when the term "complementary" is used to describe a first nucleoside sequence relative to a second nucleoside sequence, it refers to the ability of an oligonucleoside containing the first nucleoside sequence to hybridize with an oligonucleoside containing the second nucleoside sequence under certain conditions and form a duplex structure, as understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C for 12 - 16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press).

[0417] As described herein, complementary sequences within a nucleic acid (e.g., dsiRNA) include base pairing of an oligonucleoside containing a first nucleoside sequence with an oligonucleoside containing a second nucleoside sequence over the entire length of one or both nucleoside sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" or "partially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, e.g., 2, 4, or 5 mismatched base pairs, but preferably no more than 5, while maintaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibiting gene expression via the RISC pathway. In the determination of complementarity, overhangs should not be considered mismatches. For example, a nucleic acid (e.g., dsiRNA) containing an oligonucleoside of 17 nucleosides in length and another oligonucleoside of 19 nucleosides in length, where the longer oligonucleoside contains a sequence of 17 nucleosides that is fully complementary to the shorter oligonucleoside, can still be referred to as "fully complementary".

[0418] As used herein, "complementary" sequences can also include non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, or consisting entirely of non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, provided that the requirements regarding their hybridization ability as described above are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0419] The terms "complementary", "fully complementary", and "substantially / partially complementary" herein can be used to refer to base matching between the sense and antisense strands of a nucleic acid (e.g., dsiRNA), or between the antisense strand of a double-stranded nucleic acid (e.g., siRNA agent) and a target sequence.

[0420] In the present application, the second strand of the nucleic acid according to the present application is at least partially complementary to the first strand of the nucleic acid. In certain embodiments, the first and second strands of the nucleic acid according to the present application are partially complementary if they form a double-stranded region that is at least 17 base pairs in length and the double-stranded region contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0421] In certain embodiments, the first and second strands of the nucleic acid according to the present application are partially complementary if they form a double-stranded region that is 19 base pairs in length and the double-stranded region contains no more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, the first and second strands of the nucleic acid according to the present application are partially complementary if they form a double-stranded region that is 21 base pairs in length and the double-stranded region contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0422] Alternatively, the first and second strands of the nucleic acid according to the present application are partially complementary if they form a double-stranded region that is at least 17 base pairs in length, wherein at least 14, 15, 16, or 17 of the base pairs are complementary base pairs, particularly Watson-Crick base pairs.

[0423] In certain embodiments, the first and second strands of the nucleic acid according to the present application are partially complementary if they form a double-stranded region that is 19 base pairs in length, wherein at least 14, 15, 16, 17, 18, or all 19 of the base pairs are complementary base pairs, particularly Watson-Crick base pairs. In certain embodiments, the first and second strands of the nucleic acid according to the present application are partially complementary if they form a double-stranded region that is 21 base pairs in length, wherein at least 16, 17, 18, 19, 20, or all 21 of the base pairs are complementary base pairs, particularly Watson-Crick base pairs.

[0424] As used herein, a nucleic acid that is "substantially complementary" or "partially complementary" to at least a portion of messenger RNA (mRNA) refers to a nucleic acid that is substantially or partially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding a gene). In certain embodiments, the continuous portion of the mRNA is any of the sequences listed in Table 1, i.e., any of SEQ ID NO:3-42 and SEQ ID NO:443-448. For example, if a sequence is substantially or partially complementary to an uninterrupted portion of the mRNA encoding a gene of interest, the nucleic acid is complementary to at least a portion of the mRNA of the gene of interest.

[0425] Thus, in some preferred embodiments, the antisense oligonucleotides disclosed herein are fully complementary to the target gene sequence.

[0426] In other embodiments, the antisense oligonucleotides disclosed herein are substantially or partially complementary to a target RNA sequence and comprise a continuous nucleoside sequence that is at least about 80% complementary to an equivalent region of the target RNA sequence over its entire length, such as at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary or 100% complementary.

[0427] In certain embodiments, the first strand (antisense strand) of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of the RNA transcribed from the HCII gene. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of at least 17 nucleosides of the HCII mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of the HCII mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 3-22 and SEQ ID NOs: 445-446).

[0428] In certain embodiments, if the first strand (antisense strand) of the nucleic acid according to the present application comprises a continuous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of the HCII mRNA, then it is partially complementary to the continuous portion of the HCII mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 3-22 and SEQ ID NOs: 445-446). In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleoside sequence of 19 nucleosides, wherein at least 14, 15, 16, 17, 18 or all 19 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 3-22 and SEQ ID NOs: 445-446). In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleoside sequence of 23 nucleosides, wherein at least 18, 19, 20, 21, 22 or all 23 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., SEQ ID NOs: 3-22 and SEQ ID NOs: 445-446).

[0429] In certain embodiments, the first strand (antisense strand) of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of the RNA transcribed from the ZPI gene. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of at least 17 nucleotides of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleotides of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleotides of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 23-43 and SEQ ID NOs: 443-444).

[0430] In certain embodiments, if the first strand (antisense strand) of the nucleic acid according to the present application comprises a continuous nucleotide sequence of at least 17 nucleotides, wherein at least 14, 15, 16, or 17 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of the ZPI mRNA, then it is partially complementary to the continuous portion of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleotide sequence of at least 17 nucleotides, wherein at least 14, 15, 16, or 17 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 23-42 and SEQ ID NOs: 443-444). In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleotide sequence of 19 nucleotides, wherein at least 14, 15, 16, 17, 18, or all 19 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 23-42 and SEQ ID NOs: 443-444). In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleotide sequence of 23 nucleotides, wherein at least 18, 19, 20, 21, 22, or all 23 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 23-42 and SEQ ID NOs: 443-444).

[0431] In certain embodiments, the first strand (antisense strand) of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of the RNA transcribed from the B4GALT1 gene. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of at least 17 nucleotides of the B4GALT1 mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleotides of the B4GALT1 mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleotides of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 447 - 448).

[0432] In certain embodiments, if the first strand (antisense strand) of the nucleic acid according to the present application comprises a continuous nucleotide sequence of at least 17 nucleotides, wherein at least 14, 15, 16, or 17 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of the B4GALT1 mRNA, then it is partially complementary to the continuous portion of the B4GALT1 mRNA. In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleotide sequence of at least 17 nucleotides, wherein at least 14, 15, 16, or 17 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 447 - 448). In certain embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleotide sequence of 19 nucleotides, wherein at least 14, 15, 16, 17, 18, or all 19 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 447 - 448). In certain embodiments, the first strand of the nucleic acid of the present application comprises a continuous nucleotide sequence of 23 nucleotides, wherein at least 18, 19, 20, 21, 22, or all 23 nucleotides of the continuous nucleotide sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NOs: 447 - 448).

[0433] In some embodiments, the nucleic acid of the present application (e.g., siRNA) comprises a sense strand that is substantially or partially complementary to an antisense oligonucleotide, which in turn is complementary to a target gene sequence and comprises a continuous nucleotide sequence. The nucleotide sequence of the sense strand is generally at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, to the equivalent region of the nucleotide sequence of the antisense strand over its entire length.

[0434] In some embodiments, the nucleic acids (e.g., siRNA) of the present application include an antisense strand that is substantially or partially complementary to a target sequence and contains a continuous nucleoside sequence that is at least 80% complementary to the target sequence over its entire length, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.

[0435] As used herein, a "subject" is an animal, such as a mammal, including a primate (e.g., a human, a non-human primate, such as a monkey and a chimpanzee) or a non-primate or a bird that endogenously or heterologously expresses a target gene when the target gene sequence has sufficient complementarity with a nucleic acid (e.g., an siRNA agent) to facilitate target knockdown. In certain preferred embodiments, the subject is a human.

[0436] The term "treating or treatment" refers to a beneficial or desired result, including but not limited to alleviating or improving one or more symptoms associated with gene expression. "Treatment" can also mean an extended survival compared to the expected survival without treatment. Treatment can include preventing the development of complications, such as reducing liver damage in a subject with a liver infection.

[0437] As used herein, a "therapeutically effective amount" is intended to include the amount of a nucleic acid (e.g., siRNA) that, when administered to a patient to treat a subject having a disease, is sufficient to effect treatment of the disease (e.g., by alleviating, improving, or maintaining an existing disease or one or more symptoms thereof or its associated complications).

[0438] The phrase "pharmaceutically acceptable" as used herein refers to a compound, material, composition, or dosage form that is suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications and that has a reasonable benefit / risk ratio.

[0439] The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, involved in transporting or delivering a target compound from one organ or body part to another. Each carrier must be "acceptable", i.e., compatible with the other ingredients in the formulation and not injurious to the subject being treated.

[0440] When enumerating numerical values or ranges of parameters, it is intended that the numerical values or ranges between the enumerated values also become part of the present application.

[0441] As used herein, the articles "a" and "an" refer to one or more (i.e., at least one) grammatical objects of the article.

[0442] As used herein, the term "comprising" means the phrase "including but not limited to" and may be used interchangeably with that phrase.

[0443] As used herein, the term "or" means the term "and / or" and may be used interchangeably with that term "and / or" unless the context clearly dictates otherwise. For example, "sense strand or antisense strand" should be understood as "sense strand or antisense strand or sense strand and antisense strand".

[0444] As used herein, the term "about" refers to within the typical tolerances in the art. For example, "about" can be understood to be about 2 standard deviations from the mean. In certain embodiments, about represents +10%. In certain embodiments, about represents +5%. When "about" appears before a series of numbers or a range, it should be understood that "about" can modify each number in that series or range.

[0445] The term "at least" before a number or a series of numbers should be understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can logically be included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least appears before a series of numbers or a range, it should be understood that "at least" can modify each number in that series or range.

[0446] As used herein, "not more than" or "less than" should be understood to mean the value adjacent to the phrase and the logically lower value or integer, down to zero, as logically derived from the context. For example, a duplex having an overhang "not more than 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "not more than" appears before a series of numbers or a range, it should be understood that "not more than" can modify each number in that series or range.

[0447] The terminal region of a strand is the last 5 nucleotides from the 5' or 3' end.

[0448] Those skilled in the art can combine the various embodiments of the present application as needed.

[0449] Base-free nucleoside

[0450] In certain embodiments, the nucleic acid according to the present application has, for example, 1, for example 2, for example 3, for example 4 or more abasic nucleosides. Abasic nucleosides are modified nucleosides because they lack the base normally seen at the position 1 of the sugar moiety. Generally, there is a hydrogen at the position 1 of the sugar moiety of the abasic nucleosides present in the nucleic acid according to the present application.

[0451] The abasic nucleosides are in the terminal region of the second strand, preferably within the terminal 5 nucleosides at the end of the strand. The terminal region can be the terminal 5 nucleosides, which include the abasic nucleosides.

[0452] The second strand can include, as preferred features (all of which are specifically considered in combination unless mutually exclusive):

[0453] There are 2 or more than 2 abasic nucleosides in the terminal region of the second strand; and / or

[0454] There are 2 or more than 2 abasic nucleosides in the 5' or 3' terminal region of the second strand; and / or

[0455] There are 2 or more than 2 abasic nucleosides in the 5' or 3' terminal region of the second strand, wherein the abasic nucleosides are present in the overhangs as described herein; and / or

[0456] There are 2 or more than 2 consecutive abasic nucleosides in the terminal region of the second strand, wherein preferably one such abasic nucleoside is the terminal nucleoside; and / or

[0457] There are 2 or more than 2 consecutive abasic nucleosides in the 5' or 3' terminal region of the second strand, wherein preferably one such abasic nucleoside is the terminal nucleoside in the 5' or 3' terminal region of the second strand; and / or

[0458] An inverted internucleoside bond links at least one abasic nucleoside in the terminal region of the second strand to an adjacent base nucleoside; and / or

[0459] Inverted internucleoside bonding links at least one abasic nucleoside in the 5' or 3' terminal region of the second strand to an adjacent base nucleoside; and / or

[0460] The abasic nucleoside is the penultimate nucleoside, which is linked by an inverted bond to the nucleoside of the non-terminal nucleoside (referred to herein as the antepenultimate nucleoside); and / or

[0461] When reading the strand in the direction towards the end containing the terminal nucleoside, the abasic nucleoside is 2 terminal nucleosides linked by a 5'-3' bond;

[0462] When reading the strand in the direction towards the end containing the terminal nucleoside, the abasic nucleoside is 2 terminal nucleosides linked by a 3'-5' bond;

[0463] The abasic nucleosides are in the terminal two positions, wherein the penultimate nucleoside is linked to the antepenultimate nucleoside by an inverted bond, and wherein the inverted bond is a 5-5' inverted bond or a 3'-3' inverted bond;

[0464] The abasic nucleosides are in the terminal two positions, wherein the penultimate nucleoside is linked to the antepenultimate nucleoside by an inverted bond, and wherein

[0465] (1) the inverted bond is a 5-5' inverted bond, and when read towards the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'5'; or

[0466] (2) the inverted bond is a 3-3' inverted bond, and when read towards the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 5'3'.

[0467] Preferably, there are abasic nucleosides at the end of the second strand.

[0468] Preferably, in the terminal region of the second strand, preferably there are 2 or at least 2 abasic nucleosides at the end and the penultimate position.

[0469] Preferably, 2 or more abasic nucleosides are consecutive, for example all abasic nucleosides can be consecutive. For example, the terminal 1 or 2 or 3 or 4 nucleosides can be abasic nucleosides.

[0470] The abasic nucleosides can also be linked to adjacent nucleosides by 5'-3' phosphodiester bonds or inverted bonds, unless there is only 1 abasic nucleoside at the end, in which case it has an inverted bond with the adjacent nucleoside.

[0471] The inverted bond (which can also be referred to as an inverted linkage and is also common in the art) comprises a 5'-5', 3'3', 3'-2' or 2'-3' phosphodiester bond between adjacent sugar moieties of the nucleosides.

[0472] The non-terminal abasic nucleosides have 2 phosphodiester bonds, one for each adjacent nucleoside, and these bonds can be inverted bonds, or can be 5'-3 phosphodiester bonds, or can be one of each type of bond.

[0473] Preferred embodiments comprise 2 abasic nucleosides at the end and the penultimate position of the second strand, and wherein the inverted internucleoside bond is between the penultimate (abasic) nucleoside and the antepenultimate nucleoside.

[0474] Preferably, there are 2 abasic nucleosides at the end and the penultimate position of the second strand, and the penultimate nucleoside is linked to the second last nucleoside by an inverted internucleoside bond and is linked to the terminal nucleoside by a 5'-3' or 3'-5' phosphodiester bond (read along the direction of the molecular end).

[0475] Preferably, the nucleic acid according to the present application comprises one or more abasic nucleosides, optionally, wherein one or more abasic nucleosides are in the terminal region of the second strand, and / or wherein at least one abasic nucleoside is linked to an adjacent base nucleoside by a reverse internucleoside bond.

[0476] Typically, the second strand comprises 2 consecutive abasic nucleosides in its 5' terminal region, wherein one such abasic nucleoside is the terminal nucleoside of the 5' terminal region of the second strand, and the other abasic nucleoside is the penultimate nucleoside of the 5' terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the adjacent first base nucleoside in the adjacent 5'-proximal terminal region by a reverse internucleoside bond; (b) the reverse bond is a 5-5' reverse bond; and (c) when read towards the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'5'. More typically, (i) the first and second strands each have a length of 23 nucleosides; (ii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 5'-proximal terminal region of the second strand, wherein the first phosphorothioate internucleoside bond is present between the adjacent first base nucleoside and the adjacent second base nucleoside in the 5'-proximal terminal region of the second strand as in (a), and the second phosphorothioate internucleoside bond is present between the adjacent second base nucleoside and the adjacent third base nucleoside in the 5'-proximal terminal region of the second strand; (iii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 5' and 3' terminal regions of the first strand, whereby each terminal nucleoside in the 5' and 3' terminal regions of the first strand is respectively linked by phosphorothioate internucleoside bonding to its respective 5' and 3' adjacent penultimate nucleosides, and each of the former 5' and 3' penultimate nucleosides is linked by phosphorothioate internucleoside bond to its respective 5' and 3' adjacent antepenultimate nucleosides; and (iv) the second strand of the nucleic acid is directly or indirectly coupled to one or more ligand moieties in the 3' terminal region of the second strand.

[0477] Alternatively, the second strand comprises two consecutive abasic nucleosides, preferably in an overhang in the 3'-terminal region of the second strand, wherein one such abasic nucleoside is the terminal nucleoside of the 3'-terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside of the 3'-terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to an adjacent first base nucleoside in the adjacent 3'-proximal region by a reverse internucleoside bond; (b) the reverse bond is a 3-3' reverse bond; and (c) when read towards the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 5'-3'. More typically, (i) the first and second strands each have a length of 23 nucleosides; (ii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 3'-proximal region of the second strand, wherein the first phosphorothioate internucleoside bond is between the adjacent first base nucleoside and the adjacent second base nucleoside in the 3'-proximal region of the second strand, and the second phosphorothioate internucleoside bond is between the adjacent second base nucleoside and the adjacent third base nucleoside in the 3'-proximal region of the second strand; (iii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 5'- and 3'-terminal regions of the first strand, whereby each terminal nucleoside in the 5'- and 3'-terminal regions of the first strand is respectively linked to its 5'- and 3'-adjacent penultimate nucleoside by a phosphorothioate internucleoside bond, and each 5'- and 3'-penultimate nucleoside is linked to its 5'- and 3'-adjacent antepenultimate nucleoside by a phosphorothioate internucleoside bond; and (iv) the second strand of the nucleic acid is directly or indirectly coupled to one or more ligand moieties in the 5'-terminal region of the second strand.

[0478] Examples of structures are as follows (wherein the specific RNA nucleosides shown are not limiting and can be any RNA nucleoside):

[0479] A 3-3' reverse bond (and also shows the 5'-3' direction of the last phosphodiester bond between two abasic molecules read towards the end of the molecule)

[0480]

[0481] B Illustrates a 5-5' reverse bond (and also shows the 3'-5' direction of the last phosphodiester bond between two abasic molecules read towards the end of the molecule)

[0482]

[0483] One or more abasic nucleosides present in the nucleic acid are provided in the presence of one or more inverted internucleoside linkages (i.e., 5'-5' or 3'-3' inverted internucleoside linkages). The inverted linkages occur due to a change in the orientation of the adjacent nucleoside sugars such that the sugars have a 3'-5' orientation rather than the conventional 5'-3' orientation (referring to the numbering of the ring atoms on the nucleoside sugar). One or more abasic nucleosides present in the nucleic acids of the present application preferably include such inverted nucleoside sugars.

[0484] In the case where the terminal nucleoside has an inverted orientation, this will result in an "inverted" terminal configuration for the entire nucleic acid. Although some of the structures drawn and referred to herein use the conventional 5'-3' orientation representation (referring to the numbering of the ring atoms on the nucleoside sugar), it should be understood that the presence of a terminal nucleoside with a change in orientation and a proximal 3'-3' inverted linkage will result in the nucleic acid having an overall 5'-5' end structure (i.e., the conventional 3' terminal nucleoside becomes the 5' terminal nucleoside). Alternatively, it should be understood that the presence of a terminal nucleoside with a change in orientation and a proximal 5'-5' inverted linkage will result in the nucleic acid having an overall 3'-3' end structure.

[0485] A proximal 3'-3' or 5'-5' inverted linkage as described herein can comprise an inverted linkage directly adjacent / linked to a terminal nucleoside with an inverted orientation, such as a single terminal nucleoside with an inverted orientation. Alternatively, a proximal 3'-3' or 5'-5' inverted linkage as described herein can comprise an inverted linkage of two or more adjacent nucleosides with an inverted orientation, such as two or more terminal region nucleosides with an inverted orientation, such as the terminal and the penultimate nucleoside. In this way, the inverted linkage can be linked to the penultimate nucleoside with an inverted orientation. Although one of ordinary skill in the art (addressee) will understand that the inverted orientation as described above can result in the nucleic acid molecule having an overall 3'-3' or 5'-5' end structure as described herein, it should also be understood that due to the presence of one or more additional inverted linkages and / or nucleosides with an inverted orientation, the overall nucleic acid can have a 3'-5' end structure corresponding to the conventionally positioned 5' / 3' ends.

[0486] In one aspect, the nucleic acid can have a 3'-3' inverted linkage and the terminal sugar moiety can comprise a 5' OH at the 5' position of the terminal sugar rather than a 5' phosphate group.

[0487] Thus, one of ordinary skill in the art will clearly understand that 5'-5', 3'-3', and 3'-5' (read along the terminal direction) end variants of the more conventional 5'-3' structures (referring to the numbering of the ring atoms on the terminal nucleoside sugar) drawn herein are included within the scope of the present disclosure where there are inverted linkages.

[0488] In cases where, for example, reverse internucleoside bonds and / or one or more nucleosides have an inverted orientation resulting in inverted ends, and where the relative position of the bonds (e.g., bonds to the linker) or the position of internal features (e.g., modified nucleosides) are defined relative to the 5' or 3' end of the nucleic acid, the 5' or 3' end is the conventional 5' or 3' end that would be present if there were no reverse bonds, and where the conventional 5' or 3' end is determined by considering the directionality of the majority of internal nucleoside bonds and / or nucleoside orientation within the nucleic acid. From these internal bonds and / or nucleoside orientations, it can be determined which ends of the nucleic acid would constitute the conventional 5' and 3' ends of the molecule in the absence of reverse bonds (referencing the ring atom numbering on the terminal nucleoside sugar).

[0489] For example, in the structure shown below, the first 2 positions at the 5' end have abasic residues. When the terminal nucleoside has an inverted orientation, the 5' end shown in the figure below (which is the conventional 5' end) can actually contain a 3' OH according to the inverted nucleoside at the terminal position. Nevertheless, when read in the standard 5'[PO4] to 3'[OH] direction of the nucleic acid molecule (referencing the ring atom numbering on the nucleoside sugar), most of the molecule will contain conventional internucleoside bonds from the 3' OH of the sugar to the 5' phosphate of the next sugar, which can be used to determine the conventional 5' and 3' ends that would be found in the absence of an inverted terminal configuration.

[0490] 5’A-A-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me 3’

[0491] The reverse bond is preferably located at the end of the nucleic acid (e.g., RNA) that is distal from the ligand portion of the molecule (e.g., the portion containing GalNAc).

[0492] A GalNAc-siRNA construct having 5'-GalNAc on the sense strand can have a reverse bond at the other end of the sense strand.

[0493] A GalNAc-siRNA construct having 3'-GalNAc on the sense strand can have a reverse bond at the other end of the sense strand.

[0494] In certain embodiments, the present application relates to a nucleic acid for inhibiting the expression of a target gene, comprising a duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0495] wherein the second strand comprises 2 consecutive abasic nucleosides in the 5' terminal region of the second strand, where one such abasic nucleoside is the terminal nucleoside in the 5' terminal region of the second strand, and the other abasic nucleoside is the penultimate nucleoside in the 5' terminal region of the second strand, wherein:

[0496] (a) The penultimate abasic nucleoside is linked to an adjacent first base nucleoside in the adjacent 5'-proximal region by a reverse internucleoside bond;

[0497] (b) The reverse bond is a 5-5' reverse bond; and

[0498] (c) When read towards the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'-5'.

[0499] In certain embodiments, the present application relates to a nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region comprising: a first strand at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand, wherein:

[0500] (i) Preferably, each of the first strand and the second strand has a length of 23 nucleosides (the length of the second strand includes two abasic nucleosides);

[0501] (ii) The second strand contains 2 consecutive abasic nucleosides in its 5'-terminal region, one of such abasic nucleosides being the terminal nucleoside in the 5'-terminal region of the second strand, and the other abasic nucleoside being the penultimate nucleoside in the 5'-terminal region of the second strand, wherein:

[0502] (a) The penultimate abasic nucleoside is linked to an adjacent first base nucleoside in the adjacent 5'-proximal region by a reverse internucleoside bond; and

[0503] (b) The reverse linkage is a 5-5' reverse linkage; and

[0504] (c) When read towards the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'-5';

[0505] (iii) Two phosphorothioate internucleoside bonds are respectively present between three consecutive positions in the 5'-proximal region of the second strand, wherein the first phosphorothioate internucleoside bond is present between the first base nucleoside in (a) and the adjacent second base nucleoside in the 5'-proximal region of the second strand, and the second phosphorothioate internucleoside bond is present between the second base nucleoside and the adjacent third base nucleoside in the 5'-proximal region of the second strand;

[0506] (iv) Phosphorothioate internucleoside linkages are present between three consecutive positions in the 5' and 3' terminal regions of the first strand respectively, whereby each terminal nucleoside at the 5' and 3' terminal regions of the first strand is linked to its respective 5' and 3' adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage, and each 5' and 3' penultimate nucleoside is linked to its respective 5' and 3' adjacent antepenultimate nucleosides by a phosphorothioate internucleoside linkage; and

[0507] (v) The second strand of the nucleic acid is directly or indirectly coupled to one or more ligand moieties in the 3' terminal region of the second strand.

[0508] In certain embodiments, the present application relates to a nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises:

[0509] a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0510] wherein the second strand comprises 2 consecutive abasic nucleosides in the 5' terminal region of the second strand, which are presented as the following 5' terminal motif

[0511]

[0512] wherein:

[0513] B represents a nucleoside base,

[0514] T represents H, OH or a 2'-ribose modification,

[0515] Z represents the remaining nucleosides of the second strand.

[0516] In certain embodiments, the present application relates to a nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0517] wherein the second strand comprises 2 consecutive abasic nucleosides in the 5' terminal region of the second strand, which are presented as the following 5' terminal motif

[0518]

[0519] wherein:

[0520] B represents a nucleoside base,

[0521] T represents H, OH or a 2'-ribose modification,

[0522] V represents O or S (preferably O),

[0523] R represents H or C 1-4 alkyl (preferably H),

[0524] Z represents the remaining nucleosides of the second strand,

[0525] more preferably the following 5'-terminal motifs

[0526]

[0527] wherein:

[0528] B represents a nucleobase,

[0529] T represents H, OH or a 2'-ribose modification,

[0530] Z represents the remaining nucleosides of the second strand.

[0531] In certain embodiments, the present application relates to a nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising:

[0532] a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0533] wherein the second strand comprises 2 consecutive abasic nucleosides in the 5'-terminal region of the second strand, which are presented as the following 5'-terminal motifs

[0534]

[0535] wherein:

[0536] B represents a nucleobase,

[0537] T represents H, OH or a 2'-ribose modification,

[0538] V represents O or S (preferably O),

[0539] R represents H or C 1-4 alkyl (preferably H),

[0540] Z comprises 11 to 26 consecutive nucleosides, preferably 15 to 21 consecutive nucleosides, more preferably 19 consecutive nucleosides,

[0541] more preferably the following 5'-terminal motifs

[0542]

[0543] wherein:

[0544] B represents a nucleobase,

[0545] T represents H, OH or a 2'-ribose modification,

[0546] Z contains 11 to 26 consecutive nucleosides, preferably 15 to 21 consecutive nucleosides, more preferably 19 consecutive nucleosides.

[0547] In some embodiments, the modification pattern of the second strand (sense strand) of the nucleic acid according to the present application comprises or consists of the following:

[0548] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, where ia represents an inverted abasic nucleoside.

[0549] In such embodiments, the second strand preferably comprises the following 5' end motif

[0550]

[0551] where:

[0552] B represents the nucleobase of the first base nucleoside in the 5' end region of the second strand,

[0553] T represents a 2'Me ribose modification,

[0554] Z represents the remaining consecutive base nucleosides of the second strand.

[0555] In such embodiments, the modification pattern of the first strand of the nucleic acid preferably comprises or consists of the following:

[0556] Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0557] In some embodiments, the modification pattern of the second strand (sense strand) of the nucleic acid according to the present application comprises or consists of the following:

[0558] ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, where ia represents an inverted abasic nucleoside.

[0559] In such embodiments, the second strand preferably comprises the following 5' end motif

[0560]

[0561] where:

[0562] B represents the nucleobase of the first base nucleoside in the 5' end region of the second strand,

[0563] T represents a 2'Me ribose modification,

[0564] Z represents the remaining consecutive base nucleosides in the second strand.

[0565] In such embodiments, the modification pattern of the first strand of the nucleic acid preferably comprises or consists of the following:

[0566] Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me.

[0567] In some embodiments, the modification pattern of the second strand (sense strand) of the nucleic acid according to the present application comprises or consists of the following:

[0568] ia–ia-Me(s)-Me(s)-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, where (s) is a phosphorothioate internucleoside bond and ia represents an inverted abasic nucleoside.

[0569] In such embodiments, the second strand preferably comprises the following 5' end motif:

[0570]

[0571] Wherein:

[0572] B represents the nucleobase of the first two base nucleosides in the 5' end region of the second strand,

[0573] T represents a 2’Me ribose modification,

[0574] V represents O or S (preferably O),

[0575] R represents H or C 1-4 alkyl (preferably H),

[0576] Z comprises 11 to 26 consecutive base nucleosides, preferably 15 to 21 consecutive base nucleosides, more preferably 19 consecutive base nucleosides,

[0577] More preferably the following 5' end motif

[0578]

[0579] Wherein:

[0580] B represents the nucleobase of the first two base nucleosides in the 5' end region of the second strand,

[0581] T represents a 2’Me ribose modification,

[0582] Z represents the remaining 19 consecutive base nucleosides of the second strand.

[0583] In such embodiments, the modification pattern of the first strand of the nucleic acid preferably comprises or consists of the following:

[0584] Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where (s) is a phosphorothioate internucleoside bond.

[0585] In some embodiments, the modification pattern of the second strand (sense strand) of the nucleic acid according to the present application comprises or consists of the following:

[0586] ia–ia-Me(s)-Me(s)-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, where (s) is a phosphorothioate internucleoside bond and ia represents an inverted abasic nucleoside.

[0587] In such embodiments, the second strand preferably comprises the following 5' end motif

[0588]

[0589] where:

[0590] B represents the nucleobase of the first two base nucleosides in the 5' end region of the second strand,

[0591] T represents a 2’Me ribose modification,

[0592] V represents O or S (preferably O),

[0593] R represents H or C 1-4 alkyl (preferably H),

[0594] Z comprises 11 to 26 consecutive base nucleosides, preferably 15 to 21 consecutive base nucleosides, more preferably 19 consecutive base nucleosides,

[0595] More preferably, the following 5' end motif

[0596]

[0597] where:

[0598] B represents the nucleobase of the first two base nucleosides in the 5' end region of the second strand,

[0599] T represents a 2’Me ribose modification,

[0600] Z represents the remaining 19 consecutive base nucleosides of the second strand.

[0601] In such embodiments, the modification pattern of the first strand of the nucleic acid preferably comprises or consists of the following:

[0602] Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where (s) is a phosphorothioate internucleoside bond.

[0603] The reverse bond is preferably located at the end of the nucleic acid (e.g., RNA) that is distal from the ligand portion of the molecule (e.g., the GalNAc-containing portion).

[0604] A GalNAc-siRNA construct having 5'-GalNAc on the sense strand can have a reverse bond at the other end of the sense strand.

[0605] A GalNAc-siRNA construct having 3'-GalNAc on the sense strand can have a reverse bond at the other end of the sense strand.

[0606] In a preferred embodiment, the modification pattern of the second strand (sense strand) of the nucleic acid according to the present application comprises or consists of the following:

[0607] ia–ia-Me(s)-Me(s)-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0608] ia–ia-Me(s)-Me(s)-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, where (s) is a phosphorothioate internucleoside bond and ia represents an inverted abasic nucleoside.

[0609] In such embodiments, the second strand preferably comprises the following 5'-terminal motif

[0610]

[0611] Where:

[0612] B represents the nucleobase of the first two base nucleosides in the 5'-terminal region of the second strand,

[0613] T represents a 2'-Me ribose modification,

[0614] V represents O or S (preferably O),

[0615] R represents H or C 1-4 alkyl (preferably H),

[0616] Z comprises 11 to 26 consecutive base nucleosides, preferably 15 to 21 consecutive base nucleosides, more preferably 19 consecutive base nucleosides,

[0617] more preferably the following 5'-end motifs

[0618]

[0619] wherein:

[0620] B represents the nucleobases of the first two base nucleosides in the 5'-end region of the second strand,

[0621] T represents a 2'-Me ribose modification,

[0622] Z represents the remaining 19 consecutive base nucleosides of the second strand.

[0623] In such embodiments, the modification pattern of the first strand of the nucleic acid preferably comprises or consists of the following:

[0624] Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where (s) is a phosphorothioate internucleoside bond, or

[0625] Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where (s) is a phosphorothioate internucleoside bond.

[0626] Nucleic acid length

[0627] In one aspect, i) the length of the first strand of the nucleic acid ranges from 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides; and / or ii) the length of the second strand of the nucleic acid ranges from 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0628] Typically, the length of the duplex region of the nucleic acid is between 17 and 30 nucleosides, more preferably 19 or 21 nucleosides in length. Similarly, the length of the complementary region between the first strand and the RNA portion transcribed from the target gene is between 17 and 30 nucleosides.

[0629] Nucleic acid modification

[0630] In certain embodiments, the nucleic acids of the present application (e.g., RNA, e.g., dsiRNA) do not contain further modifications, such as chemical modifications or conjugations known in the art and described herein.

[0631] In other preferred embodiments, the nucleic acids of the present application (e.g., RNA, e.g., dsiRNA) are further chemically modified to enhance stability or other beneficial properties.

[0632] In certain embodiments of the present application, substantially all of the nucleosides are modified.

[0633] The nucleic acids described in the present application can be synthesized or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, the content of which is incorporated herein by reference.

[0634] Modifications include, for example, end modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleosides within RNA or RNA nucleosides within DNA, inverted linkage, etc.); base modifications, such as replacement with stabilizing bases, destabilizing bases, or bases that pair with bases having an expanded library of pairing partners, conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or sugar replacement; or backbone modifications, including modification or replacement of the phosphodiester bond.

[0635] Specific examples of nucleic acids (e.g., siRNA compounds) used in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking native internucleoside linkages. Nucleic acids (e.g., RNA) having modified backbones include, but are not limited to, those having no phosphorus atoms in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified nucleic acids such as RNAs lacking phosphorus atoms in their internucleoside backbone may also be considered oligonucleosides. In some embodiments, the modified nucleic acids (e.g., siRNA) will have phosphorus atoms in their internucleoside backbone.

[0636] Modified nucleic acid backbones such as RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thiophosphoramidates, thioalkyl phosphonates, thioalkyl phosphotriesters, and boranophosphates having a normal 3'-5' bond, 2'-5' linked analogs thereof, and those having inverted polarity (wherein adjacent nucleoside units are linked 5'-3' or 5'-2'). Also included are various salt, mixed salt, and free acid forms.

[0637] Modified nucleic acids, such as RNA, may also contain one or more substituted sugar moieties. The nucleic acids described herein (e.g., siRNA, such as dsiRNA) may include at one of the following at the 2'-position: OH, F, O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted. 2'-O-methyl and 2'-F are preferred modifications.

[0638] In certain preferred embodiments, the nucleic acid comprises at least one modified nucleoside.

[0639] The nucleic acids of the present application may contain one or more modified nucleosides on the first strand and / or the second strand.

[0640] In some embodiments, substantially all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand contain modifications.

[0641] In some embodiments, all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand contain modifications.

[0642] In some embodiments, all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand contain modifications.

[0643] In one embodiment, at least one modified nucleoside is selected from the group consisting of: deoxynucleoside, 3'-terminal deoxythymidine (dT) nucleoside, 2'-O-methyl modified nucleoside (also referred to herein as 2'-Me, where Me is methoxy), 2'-fluoro modified nucleoside, 2'-deoxy modified nucleoside, locked nucleoside, unlocked nucleoside, conformationally restricted nucleoside, restricted ethyl nucleoside, abasic nucleoside, 2'-amino modified nucleoside, 2'-O-allyl modified nucleoside, 2'-C-alkyl modified nucleoside, 2'-hydroxy modified nucleoside, 2'-methoxyethyl modified nucleoside, 2'-O-alkyl modified nucleoside, morpholino nucleoside, phosphoramidate, nucleoside containing a non-natural base, tetrahydropyran modified nucleoside, 1,5-anhydrohexitol modified nucleoside, cyclohexenyl modified nucleoside, nucleoside containing a phosphorothioate group, nucleoside containing a methylphosphonate group, nucleoside containing a 5'-phosphate, and nucleoside containing a 5'-phosphate mimic. In another embodiment, the modified nucleoside includes a short sequence of 3'-terminal deoxy-thymidine nucleoside (dT).

[0644] Modifications on the nucleoside can preferably be selected from the following group: including but not limited to LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy and combinations thereof. In another embodiment, the modification on the nucleoside is a 2'-O-methyl ("2'-Me") or 2'-fluoro modification.

[0645] A preferred modification is a modification at the 2'-OH group of the ribose, optionally selected from 2'-Me or 2'-F modifications.

[0646] Preferred nucleic acids contain one or more modified nucleosides on the first strand and / or the second strand to form modified nucleosides, as follows:

[0647] A nucleic acid, wherein the modification is a modification at the 2'-OH group of the ribose, optionally selected from 2'-Me or 2'-F modifications.

[0648] A nucleic acid, wherein the first strand contains a 2'-F modification at any one or any combination of positions 2, 6, 14 starting from position 1 of the first strand.

[0649] A nucleic acid, wherein the second strand contains a 2'-F modification at any one or any combination of positions 7, 9, 11 starting from position 1 of the second strand.

[0650] A nucleic acid, wherein the first strand and the second strand each contain 2'-Me and 2'-F modifications.

[0651] A nucleic acid, which contains at least one heat-destabilizing modification, suitably at one or more positions from position 1 to position 9 of the first strand starting from position 1 of the first strand, and / or at one or more positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid, more preferably (S)-glycol nucleic acid.

[0652] A nucleic acid, which contains at least one heat-destabilizing modification at position 7 of the first strand starting from position 1 of the first strand.

[0653] A nucleic acid, which is a siRNA oligonucleoside, wherein the siRNA oligonucleoside contains 3 or more 2'-F modifications at positions 6 to 12 of the second strand starting from position 1 of the second strand, for example, contains 4, 5, 6 or 7 2'-F modifications at positions 6 to 12 of the second strand.

[0654] A nucleic acid, which is an siRNA oligonucleotide, wherein the second strand comprises at least 3, such as 4, 5 or 6 2'-Me modifications at positions 1 to 6 of the second strand, starting from position 1 of the second strand.

[0655] A nucleic acid, which is an siRNA oligonucleotide, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3'-terminal region, preferably including the 3'-terminal region, or at least the terminal nucleoside within 1 or 2 nucleosides of the terminal nucleoside in the 3'-terminal region.

[0656] A nucleic acid, which is an siRNA oligonucleotide, wherein the first strand comprises 7 consecutive 2'-Me modifications in the 3'-terminal region, preferably including the terminal nucleoside of the 3'-terminal region.

[0657] A nucleic acid, which is an siRNA oligonucleotide, wherein each of the first strand and the second strand comprises an alternating modification pattern, preferably a fully alternating modification pattern along the entire length of each of the first strand and the second strand, wherein the nucleosides of the first strand are modified by: (i) 2'-Me modification on odd-numbered nucleosides starting from position 1 of the first strand, and (ii) 2'-F modification on even-numbered nucleosides starting from position 1 of the first strand, and the nucleosides of the second strand are modified by: (i) 2'-F modification on odd-numbered nucleosides starting from position 1 of the second strand, and (ii) 2'-Me modification on even-numbered nucleosides starting from position 1 of the second strand. Generally, such a fully alternating modification pattern is present in blunt-ended oligonucleotides, wherein the lengths of both the first strand and the second strand are 19 nucleosides.

[0658] Position 1 of the first or second strand is the nucleoside closest to the end of the nucleic acid (ignoring any abasic nucleosides), and is connected to the adjacent nucleoside (at position 2) by an internal 3'-to-5' bond, referring to the bond between the backbone sugar moieties and reading in the direction away from the end of the molecule.

[0659] Thus, it can be seen that "the position 1 of the sense strand" is the nucleoside closest to the 5'-end at the conventional 5'-end of the sense strand (excluding abasic nucleosides). Generally, the nucleoside at position 1 of the sense strand corresponds to the 5'-nucleoside of the selected target nucleic acid sequence, and more generally, the sense strand has nucleosides equivalent to those of the target nucleic acid sequence starting from this position 1 of the sense strand, while also allowing acceptable mismatches between sequences.

[0660] As used herein, "the position 1 of the antisense strand" is the nucleoside closest to the 5'-end at the conventional 5'-end of the antisense strand (excluding abasic nucleosides). As mentioned above, there will be a complementary region between the sense strand and the antisense strand, such that the antisense strand also has a region complementary to the above-mentioned target nucleic acid sequence.

[0661] In certain embodiments, the nucleic acid (e.g., an siRNA agent) further comprises at least one phosphorothioate or methylphosphonate internucleoside bond. For example, the phosphorothioate or methylphosphonate internucleoside bond can be at the 3'-end or terminal region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands (i.e., the sense strand and the antisense strand).

[0662] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside bond is at the 5'-end or terminal region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands (i.e., the sense strand and the antisense strand).

[0663] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside bond is at the 5'- and 3'-ends or in the terminal region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands (i.e., the sense strand and the antisense strand).

[0664] Any nucleic acid can contain one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS internucleoside bonds at the ends of the strand.

[0665] At least one oligoribonucleotide strand preferably contains at least two consecutive phosphorothioate modifications among the last 3 nucleosides of the oligonucleotide.

[0666] Accordingly, the present application also relates to: a nucleic acid disclosed herein that contains phosphorothioate internucleoside bonds respectively between at least two or three consecutive positions, such as at the 5' and / or 3' terminal regions and / or near-terminal regions of the second strand, wherein the near-terminal region preferably adjoins the terminal region where one or more abasic nucleosides of the second strand are located.

[0667] A nucleic acid disclosed herein that contains phosphorothioate internucleoside bonds respectively between at least two or three consecutive positions in the 5' and / or 3' terminal regions of the first strand, wherein preferably the terminal positions in the 5' and / or 3' terminal regions of the first strand are connected to their adjacent positions by phosphorothioate internucleoside bonds.

[0668] The nucleic acid strand can be RNA containing a phosphorothioate internucleoside bond between three nucleosides, and the three nucleosides are adjacent to two abasic nucleosides at the ends.

[0669] Preferred nucleic acids are double-stranded RNAs that contain 2 adjacent abasic nucleosides at the 5'-end of the second strand and a ligand portion containing one or more GalNAc ligand moieties at the other 3'-end of the second strand. Further preferably, the same nucleic acid can further contain a phosphorothioate bond between the nucleotides at positions 3-4 and positions 4-5 read from position 1 of the second strand. Further preferably, the same nucleic acid can further contain 2'-F modifications at positions 7, 9, 11 of the second strand.

[0670] Preferred modifications are as follows.

[0671] A nucleic acid, wherein the modified nucleoside of the second strand has a modification pattern (5'-3') according to any one of the following:

[0672] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0673] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0674] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0675] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0676] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me.

[0677] A nucleic acid, wherein the modified nucleoside of the second strand has a modification pattern (5'-3') according to any one of the following:

[0678] Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0679] Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0680] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0681] Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0682] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0683] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me(s)Me(s), or

[0684] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), or

[0685] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), or

[0686] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), or

[0687] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)-Me(s)Me(s),

[0688] where (s) is a phosphorothioate internucleoside bond.

[0689] A nucleic acid, wherein the modified nucleosides of the second strand have a modification pattern (5'-3') according to any one of the following:

[0690] ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0691] ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0692] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0693] ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0694] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0695] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, or

[0696] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0697] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0698] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0699] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0700] wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in two nucleoside overhangs.

[0701] A nucleic acid, wherein the modified nucleosides of the second strand have a modification pattern (5'-3') according to any one of the following:

[0702] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0703] ia–ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0704] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0705] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0706] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0707] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me(s)Me(s)ia–ia, or

[0708] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, or

[0709] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, or

[0710] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, or

[0711] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia,

[0712] wherein:

[0713] (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in two nucleoside overhangs.

[0714] A nucleic acid, wherein the modified nucleoside has the following modification pattern:

[0715] Modification pattern 1: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0716] Or modification pattern 2: Second strand (5'-3'): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0717] Or modification pattern 3: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0718] Or modification pattern 4: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0719] Or modification pattern 5: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0720] Or modification pattern 6: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me.

[0721] A nucleic acid, wherein the modified nucleoside has the following modification pattern:

[0722] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0723] Or

[0724] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0725] A nucleic acid, wherein the modified nucleoside has the following modification pattern:

[0726] Modification pattern 1: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0727] Or modification pattern 2: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0728] Or modification pattern 3: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0729] Or modification pattern 4: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0730] Or modification pattern 5: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0731] Or modification pattern 6: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0732] Where (s) is a phosphorothioate internucleoside bond.

[0733] A nucleic acid, wherein the modified nucleoside has the following modification patterns:

[0734] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0735] or

[0736] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0737] where (s) is a phosphorothioate internucleoside bond.

[0738] A nucleic acid, wherein the modified nucleosides have the following modification patterns:

[0739] Modification pattern 1: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me(s)Me(s), First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0740] Or modification pattern 2: Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0741] Or modification pattern 3: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0742] Or modification pattern 4: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0743] Or modification pattern 5: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0744] Or modification pattern 6: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s), First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0745] Where (s) is a phosphorothioate internucleoside bond.

[0746] A nucleic acid, wherein the modified nucleoside has the following modification patterns:

[0747] Modification pattern 1: Second strand (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0748] Or modification pattern 2: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0749] Or modification pattern 3: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0750] Or modification pattern 4: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0751] Or modification pattern 5: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0752] Or modification pattern 6: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me,

[0753] Wherein ia represents an inverted abasic nucleoside.

[0754] A nucleic acid, wherein the modified nucleoside has the following modification patterns:

[0755] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0756] or

[0757] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0758] wherein ia represents an inverted abasic nucleoside.

[0759] A nucleic acid, wherein the modified nucleoside has the following modification patterns:

[0760] Modification pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0761] or Modification pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0762] Or modification pattern 3: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0763] Or modification pattern 4: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0764] Or modification pattern 5: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0765] Or modification pattern 6: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me,

[0766] Wherein ia represents an inverted abasic nucleoside. When the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in two nucleoside overhangs.

[0767] A nucleic acid, wherein the modified nucleoside has the following modification pattern:

[0768] Modification pattern 1: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0769] Or modification pattern 2: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0770] Or modification pattern 3: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0771] Or modification pattern 4: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0772] Or modification pattern 5: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0773] Or modification pattern 6: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0774] Wherein:

[0775] (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside.

[0776] A nucleic acid, wherein the modified nucleoside has the following modification pattern:

[0777] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0778] Or

[0779] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0780] Wherein, (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside.

[0781] A nucleic acid, wherein the modified nucleoside has the following modification pattern:

[0782] Modification pattern 1: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me(s)Me(s)ia–ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0783] Or modification pattern 2: Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0784] Or modification pattern 3: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0785] Or modification pattern 4: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0786] Or modification pattern 5: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0787] Or modification pattern 6: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)ia–ia, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0788] Wherein: (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside. When the inverted abasic nucleoside represented by ia–ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in two nucleoside overhangs.

[0789] Particularly preferred are nucleic acids in which the modified nucleosides have the following modification patterns:

[0790] Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0791] Or

[0792] Second strand (5'-3'): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0793] Wherein (s) is a phosphorothioate nucleoside internucleoside bond and ia represents an inverted abasic nucleoside.

[0794] Coupling

[0795] Another modification of the nucleic acids (e.g., RNA, e.g., siRNA) of the present application involves linking the nucleic acid (e.g., siRNA) to one or more ligand moieties, e.g., to enhance the activity, cellular distribution, or cellular uptake of the nucleic acid (e.g., siRNA) into cells, for example.

[0796] In some embodiments, the ligand moiety can be linked to the nucleic acid, e.g., siRNA oligonucleotide, via a cleavable or non-cleavable linker. The term "linker" or "linking group" refers to an organic moiety that links two parts of a compound, e.g., covalently links two parts of a compound.

[0797] The ligand can be linked to the 3'- or 5'-end of the sense strand.

[0798] The ligand is preferably coupled to the 3'-end of the sense strand of the nucleic acid (e.g., siRNA agent).

[0799] Accordingly, in another aspect, the present application relates to a conjugate for inhibiting the expression of a target gene in a cell, the conjugate comprising a nucleic acid moiety and one or more ligand moieties, the nucleic acid moiety comprising a nucleic acid as disclosed herein.

[0800] In one aspect, the second strand of the nucleic acid is directly or indirectly (e.g., via a linker) conjugated to one or more ligand moieties, wherein the ligand moiety is generally present in the terminal region of the second strand, preferably in its 3'-terminal region.

[0801] In certain embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative linked to a nucleic acid (e.g., dsiRNA) via a linker.

[0802] Accordingly, the present application relates to a conjugate, wherein the ligand moiety comprises:

[0803] i) one or more GalNAc ligands; and / or

[0804] ii) one or more GalNAc ligand derivatives; and / or

[0805] iii) one or more GalNAc ligands conjugated to the nucleic acid via a linker.

[0806] The GalNAc ligand may be directly or indirectly conjugated to the 5'- or 3'-terminal region of the second strand of the nucleic acid, preferably to its 3'-terminal region.

[0807] GalNAc ligands are well known in the art and are described, inter alia, in EP3775207A1.

[0808] In some embodiments, the GalNAc ligand is included in Figures 1 to 4 or Figure 5 any of the linkers shown in (Formula XI), wherein the "oligonucleotide" may be any nucleic acid disclosed herein. Thus, the "oligonucleotide" may contain other linkages in addition to phosphodiester linkages, such as one or more phosphorothioate linkages. Preferably, the nucleic acid according to the present application is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand via a phosphodiester linkage, more preferably to the 3'-terminal region of the second strand.

[0809] In some embodiments, the GalNAc ligand is included in Figure 3 the linker shown, wherein the "oligonucleotide" may be any nucleic acid disclosed herein. Thus, the "oligonucleotide" may contain other linkages in addition to phosphodiester linkages, such as one or more phosphorothioate linkages. Preferably, the nucleic acid according to the present application is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand via a phosphodiester linkage, more preferably to the 3'-terminal region of the second strand.

[0810] In some embodiments, the GalNAc ligand is included in Figure 5 (Formula XI) where "oligonucleotide" can be any nucleic acid disclosed herein. Thus, the "oligonucleotide" can contain other linkages in addition to phosphodiester linkages, such as one or more phosphorothioate linkages. Preferably, the nucleic acid according to the present application is a double-stranded oligonucleotide as defined herein, and the linker is coupled to the second strand via a phosphodiester bond, more preferably to the 3'-terminal region of the second strand.

[0811] In some embodiments, the GalNAc ligand is included in Figures 1 to 4 or Figure 5 (either of the linkers shown in Formula XI), where "oligonucleotide" refers to the nucleic acid according to the present application, and the nucleic acid according to the present application includes a modified second strand having the following modification pattern (5'-3'):

[0812] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me

[0813] or

[0814] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0815] where (s) is a phosphorothioate internucleoside bond and ia represents an inverted abasic nucleoside,

[0816] preferably where the linker is coupled to the 3'-terminal region of the second strand via a phosphodiester.

[0817] In some embodiments, the GalNAc ligand is included in Figure 3 the linker shown, where "oligonucleotide" refers to the nucleic acid according to the present application, and the nucleic acid according to the present application includes a modified second strand having the following modification pattern (5'-3'):

[0818] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me

[0819] or

[0820] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0821] where (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside,

[0822] preferably where the linker is coupled to the 3'-terminal region of the second strand via a phosphodiester bond.

[0823] In some embodiments, the GalNAc ligand is included in Figure 5 (Formula XI) shown linker, where "oligonucleotide" refers to the nucleic acid according to the present application, wherein the nucleic acid according to the present application includes a modified second strand having the following modification pattern (5'-3'):

[0824] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me

[0825] or

[0826] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0827] where (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside,

[0828] preferably where the linker is coupled to the 3'-terminal region of the second strand via a phosphodiester bond.

[0829] In some embodiments, the GalNAc ligand is included in Figures 1 to 4 or Figure 5 either linker shown in (Formula XI), where "oligonucleotide" refers to the nucleic acid according to the present application, wherein the nucleic acid according to the present application includes a modified second strand having the following modification pattern (5'-3'):

[0830] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me

[0831] or

[0832] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0833] where (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside,

[0834] and where the second strand has the following structure:

[0835]

[0836] where:

[0837] T represents a 2’Me ribose modification,

[0838] B represents the nucleobase of the first two base nucleosides in the 5’ terminal region of the second strand, and

[0839] Z represents the remaining 19 consecutive base nucleosides of the second strand.

[0840] In some embodiments, the GalNAc ligand is included in the Figure 3 indicated linker, where “oligonucleotide” refers to a nucleic acid according to the present application, where the nucleic acid according to the present application includes a modified second strand having the following modification pattern (5’-3’):

[0841] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me

[0842] or

[0843] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0844] where (s) is a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside,

[0845] and where the second strand has the following structure:

[0846]

[0847] where:

[0848] T represents a 2’Me ribose modification,

[0849] B represents the nucleobase of the first two base nucleosides in the 5’ terminal region of the second strand, and

[0850] Z represents the remaining 19 consecutive base nucleosides of the second strand.

[0851] In some embodiments, the GalNAc ligand is included in Figure 5 (the linker shown in formula XI), wherein "oligonucleotide" refers to the nucleic acid of the present application, and the nucleic acid of the present application comprises a modified second strand having the following modification pattern (5'-3'):

[0852] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me

[0853] or

[0854] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0855] wherein (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside,

[0856] and wherein the second strand has the following structure:

[0857]

[0858] wherein:

[0859] T represents a 2’Me ribose modification,

[0860] B represents the nucleobase of the first two base nucleosides in the 5’ terminal region of the second strand, and

[0861] Z represents the remaining 19 consecutive base nucleosides of the second strand.

[0862] The vector and the cell

[0863] In one aspect, the present application provides a cell containing a nucleic acid, such as the inhibitory RNA [RNAi] described herein.

[0864] In one aspect, the present application provides a cell containing the vector described herein.

[0865] Pharmaceutically acceptable composition

[0866] In one aspect, the present application provides a pharmaceutical composition for inhibiting the expression of a target gene, the composition comprising the nucleic acid disclosed herein.

[0867] The pharmaceutically acceptable composition may comprise excipients and / or carriers.

[0868] Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffering solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) fillers such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (22) other non-toxic and compatible substances for pharmaceutical formulations.

[0869] Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethyl cellulose, etc.); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.); lubricants (such as magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (such as starch, sodium carboxymethyl starch, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

[0870] Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration and not reacting detrimentally with the nucleic acid can also be used to formulate the compositions of the present application. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxyethyl cellulose, polyvinylpyrrolidone, etc.

[0871] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as ethanol, or nucleic acid solutions in liquid or solid oil bases. The solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration and not reacting detrimentally with the nucleic acid can be used.

[0872] In one embodiment, the nucleic acid or composition is administered in a non-buffered solution. In certain embodiments, the non-buffered solution is saline or water. In other embodiments, the nucleic acid (e.g., an siRNA agent) is administered in a buffered solution. In such embodiments, the buffered solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffered solution can be phosphate buffered saline (PBS).

[0873] Dose

[0874] The pharmaceutical compositions of the present application can be administered in a dose sufficient to inhibit gene expression. Generally, a suitable dose of the nucleic acid (e.g., siRNA) of the present application ranges from about 0.001 to about 200.0 milligrams per kilogram of body weight per day in a recipient, typically in the range of about 1 to 50 milligrams per kilogram of body weight per day. Generally, a suitable dose of the nucleic acid (e.g., siRNA) of the present application ranges from about 0.1 mg / kg to about 5.0 mg / kg, for example, about 0.3 mg / kg and about 3.0 mg / kg.

[0875] A repeated dose regimen can include the periodic administration of a therapeutically effective amount of the nucleic acid (e.g., siRNA), such as once every other day or once a year. In certain embodiments, the administration of the nucleic acid (e.g., siRNA) is from about once a month to about once every three months (i.e., about once every quarter).

[0876] In various embodiments, the nucleic acid (e.g., an siRNA agent) is administered in a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the nucleic acid (e.g., an siRNA agent) is administered in a dose of about 10 mg / kg to about 30 mg / kg. In certain embodiments, the nucleic acid (e.g., an siRNA agent) is administered in a dose selected from: about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg. In certain embodiments, the nucleic acid (e.g., the agent) is administered in a dose of about 0.1 mg / kg to about 5.0 mg / kg once a week, once a month, once every two months, or once every three months (i.e., once every quarter). In certain embodiments, the nucleic acid (e.g., an siRNA agent) is administered to a subject once a week. In certain embodiments, the nucleic acid (e.g., an siRNA agent) is administered to a subject once a month. In certain embodiments, the nucleic acid (e.g., an siRNA agent) is administered once every three months (i.e., once every quarter).

[0877] After an initial treatment regimen, treatment can be administered less frequently. For example, after once a week or once every two weeks for three months, it can be repeated once a month for six months or a year; or longer.

[0878] The pharmaceutical composition can be administered once daily, or in two, three or more sub-doses at appropriate intervals during the day, or even by continuous infusion or via a controlled release formulation. In this case, the nucleic acid (e.g., siRNA) contained in each sub-dose must be correspondingly smaller to achieve the total daily dose. The dose units can also be combined for delivery over several days, for example using a conventional sustained release formulation, which can provide a sustained release of the nucleic acid (e.g., siRNA) over several days. Sustained release formulations are well known in the art and are particularly suitable for delivering agents to specific sites, such as can be used in conjunction with the agents of the present application. In this embodiment, the dose unit contains a corresponding multiple of the daily dose.

[0879] In other embodiments, a single dose of the pharmaceutical composition can be long-acting such that subsequent doses are administered at intervals of no more than 3, 4 or 5 days, or at intervals of no more than 1, 2, 3 or 4 weeks. In some embodiments of the present application, a single dose of the pharmaceutical composition of the present application is administered once a week. In other embodiments of the present application, a single dose of the pharmaceutical composition of the present application is administered once every two months. In certain embodiments, the siRNA is administered approximately once a month to approximately once a quarter (i.e., approximately once every three months), or even once every 6 months or 12 months.

[0880] The effective dose and in vivo half-life of an individual nucleic acid (e.g., siRNA) covered by the present application can be estimated using conventional methodologies or in vivo testing based on the use of suitable animal models, as is well known in the art.

[0881] The pharmaceutical composition of the present application can be administered in a variety of ways, depending on whether local or systemic treatment is required and depending on the area to be treated. Administration can be local (e.g., via a transdermal patch), pulmonary (e.g., via inhalation or insufflation of a powder or aerosol, including via a nebulizer), intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implant device; or intracranial, e.g., via intrathecal, intracerebroventricular or parenchymal administration. In certain preferred embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

[0882] In one embodiment, a nucleic acid (e.g., a reagent) is administered subcutaneously to a subject.

[0883] The nucleic acid (e.g., siRNA) can be delivered in a manner that targets a specific tissue (e.g., especially hepatocytes).

[0884] Methods for inhibiting the expression of target genes

[0885] The present application also provides a method for inhibiting the expression of a target gene in a cell. The method comprises contacting the cell with a nucleic acid of the present application (e.g., an siRNA agent, such as a double-stranded siRNA agent) in an amount effective to inhibit the expression of the target gene in the cell, thereby inhibiting the expression of the target gene in the cell.

[0886] The contacting of the cell with the nucleic acid (e.g., siRNA, such as a double-stranded siRNA agent) can be carried out in vitro or in vivo. Contacting the cell with the nucleic acid in vivo, for example, comprises contacting the cell or a group of cells in a subject (e.g., a human subject) with the nucleic acid (e.g., siRNA). The in vitro and in vivo methods of contacting the cell can also be combined. As described above, contacting the cell can be direct or indirect.

[0887] In addition, contacting the cell can be achieved through a targeting ligand moiety, which comprises any ligand moiety described herein or known in the art. In a preferred embodiment, the targeting ligand moiety is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to a site of interest.

[0888] As used herein, the terms "inhibit" and "reduce", "silence", "downregulate", "suppress" and other similar terms are used interchangeably and include any level of inhibition.

[0889] In some embodiments of the method of the present application, the expression of the target gene is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or inhibited to below the determined detection level, preferably when determined by qPCR as described herein and / or when siRNA is introduced into the target cell by transfection. In certain embodiments, the method comprises a clinically relevant inhibition of target gene expression, e.g., as demonstrated by clinically relevant outcomes after treating a subject with an agent to reduce gene expression.

[0890] In some embodiments, when the nucleic acid of the present application is transfected into a cell, its IC50 value for inhibiting the expression of the target gene is less than 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM, preferably determined by qPCR, more preferably determined by reverse transcriptase (RT)-qPCR as described herein.

[0891] In a preferred embodiment, when the nucleic acid of the present application is transfected into cells, its IC50 value for inhibiting the expression of the target gene is less than 2000 pM. In a more preferred embodiment, when the nucleic acid of the present application is transfected into cells, its IC50 value for inhibiting the expression of the target gene is less than 1000 pM. In a more preferred embodiment, when the nucleic acid of the present application is transfected into cells, its IC50 value for inhibiting the expression of the target gene is less than 500 pM. In the most preferred embodiment, when the nucleic acid of the present application is transfected into cells, its IC50 value for inhibiting the expression of the target gene is less than 100 pM.

[0892] The inhibition of the target gene can be quantified by the following method:

[0893] Huh7 cells (a human hepatocyte-derived cell line obtained from the JCRB cell bank) can be cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS in an atmosphere of 37 °C and 5% CO2. The cells can then be transfected with siRNA duplexes targeting the mRNA transcribed from the target gene or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 487), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 486)), using 10x3-fold serial dilutions with a final duplex concentration ranging from 20 nM to 1 pM. Transfection can be carried out by adding 9.7 μL Opti-MEM (ThermoFisher) and 0.3 μL Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture can be incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells can be incubated at 37 °C / 5% CO2 for 24 hours before total RNA is purified using the RNeasy 96 Kit (Qiagen). Each duplex can be tested by transfection in replicate wells in a single experiment.

[0894] cDNA synthesis can be carried out using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) can be carried out using the FastStart Universal Probe Master kit (Roche) with specific primers for the target gene and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or ABI QuantStudio 7.

[0895] Replicates of cDNA from each well can be subjected to qPCR, and the average cycle threshold (Ct) can be calculated. The relative target gene expression can be calculated based on the average Ct value using the comparative Ct (ΔΔCt) method, normalized to GAPDH and relative to untreated cells. The four-parameter (variable slope) model of GraphPad Prism 9 can be used to calculate the maximum percent inhibition of target gene expression and the IC50 value.

[0896] Alternatively or in addition, inhibition of target gene expression can be characterized by a decrease in the average relative expression of the target gene.

[0897] In some embodiments, when cells are transfected with 0.1 nM of the nucleic acid of the present application, the average relative expression of the target gene is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4, preferably determined by qPCR, more preferably determined by reverse transcriptase (RT)-qPCR, as described herein.

[0898] In some embodiments, when cells are transfected with 5 nM of the nucleic acid of the present application, the average relative expression of the target gene is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3, preferably determined by qPCR, more preferably determined by reverse transcriptase (RT)-qPCR, as described herein.

[0899] The average relative expression of the target gene can be quantified by the following method:

[0900] Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB cell bank) can be cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS under an atmosphere of 37 °C and 5% CO2. Cells can be transfected with siRNA duplexes targeting mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO:487), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO:486)), with a final duplex concentration of 5 nM and 0.1 nM. Transfection can be carried out by adding 9.7 μL of Opti-MEM (ThermoFisher) and 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture can be incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. Cells can be incubated at 37 °C / 5% CO2 for 24 hours before purifying total RNA using the RNeasy 96 kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in two independent experiments.

[0901] cDNA synthesis can be carried out using FastQuant RT (with gDNase kit (Tiangen)). Real-time quantitative PCR (qPCR) can be carried out using the FastStart Universal Probe Master Kit (Roche) with specific primers for the target gene and human GAPDH (Hs02786624_g1) on an ABIPrism 7900HT or ABI QuantStudio 7.

[0902] qPCR can be repeated for the cDNA from each well, and the average Ct can be calculated. The relative target gene expression can be calculated using the comparative Ct (ΔΔCt) method based on the average Ct value, normalized to GAPDH and relative to untreated cells.

[0903] Inhibition of target gene expression can be demonstrated by a decrease in the amount of mRNA of the target gene compared to a suitable control.

[0904] In other embodiments, inhibition of target gene expression can be evaluated based on a decrease in a parameter related to the function of gene expression (such as protein expression or signal pathway). Exemplary target genes shown herein are HCII, ZPI, and B4GALT1.

[0905] Methods for treating or preventing diseases related to target gene expression

[0906] The present application also provides a method for reducing or inhibiting the expression of a target gene in a cell using the nucleic acid (e.g., siRNA) of the present application or a composition containing the nucleic acid (e.g., siRNA) of the present application. The method includes contacting the cell with the nucleic acid (e.g., dsiRNA) of the present application and maintaining the cell for a sufficient time to obtain degradation of the mRNA transcript of the target, thereby inhibiting the expression of the target gene in the cell. The reduction of gene expression can be evaluated by any method known in the art.

[0907] In the method of the present application, the cell can be contacted in vitro or in vivo, i.e., the cell can be in a subject's body.

[0908] Cells suitable for treatment using the method of the present application can be any cell that expresses a gene of interest associated with a disease related to a hemostatic disorder (e.g., a disease related to a hemostatic disorder, such as hemophilia). Alternatively, cells suitable for treatment using the method of the present application can be any cell that expresses a gene of interest associated with diabetes or cardiovascular disease.

[0909] The in vivo method of the present application can include administering to a subject a composition containing the nucleic acid (e.g., siRNA) of the present application, wherein the nucleic acid (e.g., siRNA) includes a nucleoside sequence complementary to at least a portion of the RNA transcript of the target gene of the mammal to be treated.

[0910] The present application also provides a method of treatment for a subject in need thereof. The method of treatment of the present application includes administering to the subject a nucleic acid (e.g., siRNA) of the present application in a therapeutically effective amount, e.g., a nucleic acid (e.g., siRNA) directed against a target gene or a pharmaceutical composition containing a nucleic acid targeting a gene, said subject being, for example, a subject who can benefit from the reduction or inhibition of the expression of the target gene.

[0911] The disease to be treated can be related to a hemostatic disorder, such as a disease related to a hemostatic disorder, such as hemophilia, especially when the target gene is HCII or ZPI as disclosed herein.

[0912] Hemophilia (haemophilia or hemophilia) is mainly an inherited disease that impairs the body's ability to produce blood clots, a process required for hemostasis. This can lead to longer bleeding times, easy bruising, and an increased risk of bleeding within joints or the brain after injury in patients. Patients with mild cases may only show symptoms after an accident or during surgery. Joint bleeding (also known as haemarthrosis) can lead to permanent damage, while bleeding in the brain can cause long-term headaches, seizures, or a decreased level of consciousness.

[0913] There are mainly two types of hemophilia: hemophilia A, which occurs due to low levels of clotting factor VIII; and hemophilia B, which occurs due to low levels of clotting factor IX. They are usually inherited from parents through the X chromosome carrying a non-functional gene. New mutations rarely occur during early development or develop into hemophilia later in life due to the formation of antibodies against clotting factors. Other types include hemophilia C, which occurs due to low levels of factor XI, von Willebrand disease, which occurs due to low levels of a substance called von Willebrand factor, and parahemophilia, which occurs due to low levels of factor V. Hemophilia A, B, and C prevent the normal operation of the intrinsic pathway; this clotting pathway is necessary when the vascular endothelium is damaged. Acquired hemophilia is associated with cancer, autoimmune diseases, and pregnancy. Diagnosis is by testing the blood's clotting ability and clotting factor levels.

[0914] In certain embodiments, the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII, are suitable for treating, or are suitable for treating hemophilia A, B, and / or C. In certain embodiments, the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII, are suitable for treating, or are suitable for treating hemophilia A and / or B. In certain embodiments, the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII, are suitable for treating, or are suitable for treating acquired hemophilia. In certain embodiments, the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII, are suitable for treating, or are suitable for treating von Willebrand disease. In certain embodiments, the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII are suitable for treating, or are suitable for treating parahemophilia.

[0915] Without being bound by theory, treatment with the nucleic acids of the present application can lead to an increase in the levels of clotting factors, thereby reducing or preventing bleeding. Thus, in a preferred embodiment, treatment with the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII, can reduce or prevent bleeding events in subjects with hemophilia. In another preferred embodiment, treatment with the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII, can reduce or prevent joint bleeding in subjects with hemophilia. In certain embodiments, treatment with the nucleic acids of the present application, particularly those that inhibit the expression of ZPI or HCII, can reduce or prevent muscle or cerebral hemorrhage in subjects with hemophilia.

[0916] The disease to be treated can be diabetes, particularly when the target gene is B4GALT1 as disclosed herein.

[0917] According to the present application, as used herein, the term "diabetes" refers to a group of metabolic diseases in which a subject has high blood sugar because the body cannot produce enough insulin or because the cells do not respond to the insulin produced. There are mainly three types of diabetes: (1) Type 1 diabetes (T1D): caused by the body's inability to produce insulin and currently requires an individual to inject insulin (also known as insulin-dependent diabetes mellitus, IDDM for short, juvenile diabetes). (2) Type 2 diabetes (T2D): caused by insulin resistance, that is, the cells cannot correctly utilize insulin, sometimes accompanied by absolute insulin deficiency (formerly known as non-insulin-dependent diabetes mellitus, NIDDM for short, or adult-onset diabetes). (3) Gestational diabetes (GD): refers to high blood sugar levels in pregnant women who have never had diabetes during pregnancy. It may precede the development of T2D.

[0918] In certain embodiments, the nucleic acids according to the present application, particularly nucleic acids that inhibit B4GALT1 expression, or pharmaceutical compositions comprising said nucleic acids are used for the treatment of diabetes, preferably type 2 diabetes (T2D).

[0919] The disease to be treated can be a cardiovascular disease, particularly when the target gene is B4GALT1 as disclosed herein.

[0920] The term "cardiovascular disease" as used herein refers to any condition, disorder or disease state related to, caused by, or resulting from an abnormal structure or function of the heart or the blood vessels supplying the heart, which abnormal structure or function impairs the normal function of the heart. Cardiovascular diseases can include coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina pectoris, deep vein thrombosis, stroke, congestive heart failure or arrhythmia. In a preferred embodiment, the cardiovascular disease is coronary artery disease.

[0921] In certain embodiments, the nucleic acids according to the present application, particularly nucleic acids that inhibit B4GALT1 expression, or pharmaceutical compositions comprising said nucleic acids are used for the treatment of cardiovascular diseases, preferably coronary artery disease.

[0922] The nucleic acids of the present application (e.g., siRNA) can be administered as "naked" nucleic acids or "naked" siRNA, administered without a pharmaceutical composition. The naked nucleic acid can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, albumin, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH value and osmotic pressure of the buffer solution can be adjusted to make it suitable for administration to a subject.

[0923] Alternatively, the nucleic acid of the present application (e.g., siRNA) can be administered as a pharmaceutical composition (e.g., dsiRNA liposomal formulation).

[0924] In one embodiment, the method comprises administering a composition as described herein such that the expression of a target gene is reduced, e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the reduction in the expression of the target gene persists for an extended period of time, e.g., at least about two days, three days, four days, or longer, e.g., about one week, two weeks, three weeks, or four weeks or longer, e.g., about 1 month, 2 months, or 3 months.

[0925] A therapeutically effective amount of a nucleic acid (e.g., siRNA), e.g., about 0.01 mg / kg to about 200 mg / kg, can be administered to a subject to treat a disease associated with a hemostatic disorder, e.g., a disease associated with a hemostatic disorder (e.g., hemophilia), or to treat diabetes or to treat cardiovascular disease.

[0926] The nucleic acid (e.g., siRNA) can be administered intravenously by infusion over a period of time on a regular basis. In certain embodiments, after an initial treatment regimen, the treatment can be administered at a lower frequency. Administering siRNA can reduce the level of the gene product of the target gene (e.g., in a patient's cells or tissues) by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay used. In certain embodiments, the administration results in a clinical stabilization or preferably a clinically relevant reduction in at least one sign or symptom of the target gene-related disease.

[0927] Alternatively, the nucleic acid (e.g., siRNA) can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver the desired daily dose of the nucleic acid (e.g., siRNA) to the subject. The injections can be repeated over a period of time. The administration can be repeated regularly. In certain embodiments, after an initial treatment regimen, the treatment can be administered at a lower frequency. The repeated dose regimen can include regular administration of a therapeutically effective amount of the nucleic acid, e.g., every other day or once a year. In certain embodiments, the nucleic acid is administered about once a month to once a quarter (i.e., about once every three months).

[0928] In one aspect, the nucleic acids disclosed herein can be nucleic acids as defined in Items 1 to 45 below:

[0929] 1. A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0930] wherein the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'):

[0931] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0932] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0933] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me.

[0934] 2. A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0935] wherein the nucleosides of the second strand have the following 2'-sugar and bond modification pattern (5'-3'):

[0936] Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0937] Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0938] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0939] Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0940] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or

[0941] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0942] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0943] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0944] where (s) is a phosphorothioate internucleoside bond.

[0945] 3. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0946] wherein the nucleosides of the second strand comprise the following 2'-sugar and abasic modification patterns (5'-3'):

[0947] ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0948] ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0949] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0950] ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0951] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, or

[0952] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0953] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0954] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0955] wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0956] 4. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand,

[0957] wherein the nucleosides of the second strand comprise the following 2'-sugar, abasic and linkage modification patterns (5'-3'):

[0958] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0959] ia–ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0960] ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0961] ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[0962] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia, or

[0963] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or

[0964] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia, or

[0965] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[0966] wherein:

[0967] (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[0968] 5. The nucleic acid according to any one of the foregoing, wherein the first strand comprises a modification pattern selected from the following or any combination thereof, wherein position 1 is the 5'-terminal nucleoside of the first strand and the counting direction is 5'-3':

[0969] a 2'-F sugar modification at least at positions 2, 14, and 16, and / or

[0970] a 2'-Me sugar modification at positions 17 to 23, or the first strand comprises at least eight 2'-F sugar modifications, such as 2'-F sugar modifications at least at positions 2, 4, 6, 12, 14, 16, 18, and 20, and / or

[0971] a 2'-Me sugar modification at positions 1, 3 to 5, 10 to 13, or the first strand comprises at least eight 2'-F sugar modifications, such as 2'-F sugar modifications at least at positions 2, 4, 6, 12, 14, 16, 18, and 20, and / or

[0972] a 2'-Me sugar modification or a heat-destabilizing modification at position 7, such as a modified unlocked nucleic acid or glycol nucleic acid usually at position 7, and / or

[0973] a 2'-F sugar modification or a heat-destabilizing modification at position 6, such as a modified unlocked nucleic acid or glycol nucleic acid usually, and / or

[0974] Positions 8 and 9 can be modifications selected from 2'-Me sugar modifications and 2'-F sugar modifications, and can usually be the same 2'-sugar modification,

[0975] Thus, the first strand can generally contain the following modification patterns (5'-3'):

[0976] Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0977] where M represents a modification selected from the group consisting of: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (e.g., generally modified unlocked nucleic acid or ethylene glycol nucleic acid), which is generally present at position 6, and usually, the 2'-Me sugar modification is present at position 7,

[0978] or thus, the first strand generally contains the following modification pattern (5'-3'):

[0979] Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0980] where M1 represents a heat-labile modification, e.g., generally modified unlocked nucleic acid or ethylene glycol nucleic acid, and M2 represents a modification selected from the group consisting of 2'-Me sugar modification and 2'-F sugar modification,

[0981] or thus, the first strand generally contains the following modification pattern (5'-3'):

[0982] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0983] where M1 represents a modification selected from the group consisting of: 2'-Me sugar modification, 2'-sugar modification, and heat-destabilizing modification (e.g., generally modified unlocked nucleic acid or ethylene glycol nucleic acid), and M2 represents a modification selected from the group consisting of 2'-Me sugar modification and 2'-F sugar modification,

[0984] or thus, the first strand generally contains the following modification pattern (5'-3'):

[0985] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[0986] where M1 represents a heat-destabilizing modification, e.g., generally modified unlocked nucleic acid or ethylene glycol nucleic acid, M2 represents a modification selected from the group consisting of 2'-Me sugar modification and 2'-F sugar modification, and usually M2 can be the same 2'-sugar modification.

[0987] 6. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[0988] wherein the nucleosides of the second strand comprise the following 2'-sugar and abasic modification patterns:

[0989] Second strand (5'-3'): ia-ia-Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me

[0990] or

[0991] Position 7 on the second strand includes a sugar modification that is a 2'-Me modification, where position 1 is the 5'-terminal nucleoside of the second strand, the counting direction is 5'-3', and there are generally two inverted abasic nucleosides in the 5'-terminal region of the second strand,

[0992] and the first strand modification pattern comprises a modification pattern selected from the following or any combination thereof, where position 1 is the 5'-terminal nucleoside of the first strand, the counting direction is 5'-3':

[0993] At least 2'-F sugar modifications at positions 2, 14, and 16, and / or

[0994] 2'-Me sugar modifications at positions 17 to 23, and / or

[0995] 2'-Me sugar modifications at positions 1, 3 to 5, 10 to 13, and / or

[0996] A 2'-Me sugar modification at position 7, or a heat destabilizing modification at position 7 (e.g., generally a modified unlocked nucleic acid or glycol nucleic acid), and / or

[0997] A 2'-F sugar modification or a heat destabilizing modification at position 6 (e.g., generally a modified unlocked nucleic acid or glycol nucleic acid), and / or

[0998] Positions 8 and 9 can be modifications selected from 2'-Me sugar modifications and 2'-F sugar modifications, and can generally be the same 2'-sugar modification,

[0999] Thus, generally the first strand can comprise the following modification pattern (5'-3'):

[1000] Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1001] Where M represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid typically), which is usually present at position 6, and usually the 2'-Me sugar modification is present at position 7,

[1002] or thus usually the first strand contains the following modification pattern (5'-3'):

[1003] Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1004] where M1 represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid typically, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification,

[1005] or thus usually the first strand contains the following modification pattern (5'-3'):

[1006] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1007] where M1 represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid typically), and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification,

[1008] or thus usually the first strand contains the following modification pattern (5'-3'):

[1009] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1010] where Ml represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid typically, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification, and usually M2 can be the same 2'-sugar modification.

[1011] 7. The nucleic acid according to item 5 or 6, wherein (M)4 represents any one of the following 2'-sugar modification patterns (5'-3'):

[1012] F–Me–Me–F

[1013] Me–F–Me–F

[1014] F–Me–F–Me

[1015] F–F–F–F

[1016] Me–F–F–Me

[1017] Me–Me–F–F

[1018] F–F–Me–Me

[1019] Me–Me–Me–Me。

[1020] 8. A nucleic acid according to any one of items 5 to 7, wherein two internucleoside phosphorothioate bonds are present between three consecutive positions in the 5' and 3' terminal regions of the first strand, whereby each of the terminal nucleosides in the 5' and 3' terminal regions of the first strand is linked to its respective 5' and 3' adjacent penultimate nucleosides by an internucleoside phosphorothioate bond, and each 5' and 3' penultimate nucleoside is linked to its respective 5' and 3' adjacent antepenultimate nucleosides by an internucleoside phosphorothioate bond, and

[1021] optionally, two internucleoside phosphorothioate bonds may also be present between three consecutive positions in the 3' terminal region of the second strand, whereby the 3' terminal nucleoside is linked to the adjacent penultimate nucleoside by an internucleoside phosphorothioate bond, and the penultimate nucleoside is linked to the adjacent antepenultimate nucleoside by an internucleoside phosphorothioate bond, and / or

[1022] optionally, two internucleoside phosphorothioate bonds may also be present between three consecutive positions in the 5' terminal region of the second strand, whereby the 5' terminal nucleoside is linked to the adjacent penultimate nucleoside by an internucleoside phosphorothioate bond, and the penultimate nucleoside is linked to the adjacent antepenultimate nucleoside by an internucleoside phosphorothioate bond.

[1023] 9. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, said duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[1024] wherein the nucleosides of the second strand and the first strand have the following 2'-sugar modification patterns (5'-3'):

[1025] Modification pattern 1:

[1026] Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me,

[1027] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1028] Or modification pattern 2:

[1029] Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1030] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1031] Or modification pattern 3:

[1032] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1033] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1034] Or modification pattern 4:

[1035] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1036] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1037] Or modification pattern 5:

[1038] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1039] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[1040] 10. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[1041] wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and linkage modification patterns (5'-3'):

[1042] Modification pattern 1:

[1043] Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me,

[1044] First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1045] Or modification pattern 2:

[1046] Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1047] First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1048] Or modification pattern 3:

[1049] Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1050] First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1051] Or modification pattern 4:

[1052] Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1053] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1054] Or modification pattern 5:

[1055] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1056] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1057] Or modification pattern 6:

[1058] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1059] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1060] wherein, (s) is a phosphorothioate internucleoside linkage.

[1061] 11. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[1062] wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and linkage modification patterns (5’-3’):

[1063] Modification pattern 1:

[1064] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me,

[1065] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1066] Or modification pattern 2:

[1067] Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[1068] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1069] Or modification pattern 3:

[1070] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[1071] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1072] Or modification pattern 4:

[1073] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[1074] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1075] Or modification pattern 5:

[1076] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[1077] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1078] Or modification pattern 6:

[1079] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[1080] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1081] where (s) is a phosphorothioate internucleoside linkage.

[1082] 12. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, said duplex region comprising: a first strand at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand,

[1083] wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and abasic modification patterns (5’-3’):

[1084] Modification pattern 1:

[1085] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me,

[1086] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1087] Or modification pattern 2:

[1088] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1089] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1090] Or modification pattern 3:

[1091] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1092] First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1093] Or modification pattern 4:

[1094] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1095] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1096] Or modification pattern 5:

[1097] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1098] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[1099] Or modification pattern 6:

[1100] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me

[1101] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[1102] Wherein ia represents an inverted abasic nucleoside.

[1103] 13. A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[1104] wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar and abasic modification patterns:

[1105] Modification pattern 1:

[1106] Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia,

[1107] First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1108] Or modification pattern 2:

[1109] Second strand (5'-3'): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[1110] First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1111] Or modification pattern 3:

[1112] Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[1113] First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1114] Or modification pattern 4:

[1115] Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[1116] First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[1117] or modification pattern 5:

[1118] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[1119] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[1120] or modification pattern 6:

[1121] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia

[1122] First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me,

[1123] wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[1124] 14. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand,

[1125] wherein the nucleosides of the second strand and the first strand comprise the following 2’ sugar, abasic, and linkage modification patterns (5’-3’):

[1126] Modification pattern 1:

[1127] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me,

[1128] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1129] Or modification pattern 2:

[1130] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[1131] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1132] Or modification pattern 3:

[1133] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[1134] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1135] Or modification pattern 4:

[1136] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1137] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1138] Or modification pattern 5:

[1139] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me,

[1140] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1141] or modification pattern 6:

[1142] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me,

[1143] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1144] wherein:

[1145] (s) represents a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside.

[1146] 15. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, said duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[1147] wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar, abasic, and bond modification patterns (5’-3’):

[1148] Modification pattern 1:

[1149] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia–ia,

[1150] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1151] or modification pattern 2:

[1152] Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[1153] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1154] Or modification pattern 3:

[1155] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[1156] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1157] Or modification pattern 4:

[1158] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[1159] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1160] Or modification pattern 5:

[1161] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[1162] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1163] Or modification pattern 6:

[1164] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia,

[1165] First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[1166] wherein:

[1167] (s) is a phosphorothioate internucleoside bond,

[1168] ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[1169] 16. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand, wherein the second strand comprises:

[1170] Starting from position 1 at the 5’ end of the second strand, where position 1 is the nucleoside closest to the 5’ end excluding abasic nucleosides, position 7 on the second strand comprises a sugar modification which is a 2’-Me modification, or

[1171] the second strand comprises the following modification pattern:

[1172] Second strand (5’-3’): Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me and wherein the first strand comprises a modification pattern selected from the following or any combination thereof, where position 1 is the 5’ end nucleoside of the first strand and the counting direction is 5’-3’:

[1173] 2’-F sugar modifications at least at positions 2, 14 and 16, and / or

[1174] 2’-Me sugar modifications at positions 17 to 23, and / or

[1175] 2’-Me sugar modifications at positions 1, 3 to 5, 10 to 13, and / or

[1176] a 2’-Me sugar modification at position 7 or a heat destabilizing modification at position 7, such as a modified unlocked nucleic acid or glycol nucleic acid, and / or

[1177] a 2’-F sugar modification or a heat destabilizing modification at position 6, such as a modified unlocked nucleic acid or glycol nucleic acid, and / or

[1178] Positions 8 and 9 can be modifications selected from 2'-Me sugar modifications and 2'-F sugar modifications, and can generally be the same 2'-sugar modification.

[1179] Thus, generally the first strand can contain the following modification patterns (5'-3'):

[1180] Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (e.g., generally modified unlocked nucleic acid or glycol nucleic acid), which is usually present at position 6, and generally the 2'-Me sugar modification is present at position 7.

[1181] Or thus, generally the first strand contains the following modification patterns (5'-3'):

[1182] Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, e.g., generally a modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from the following: 2'-Me sugar modification and 2'-F sugar modification.

[1183] Or thus, generally the first strand contains the following modification patterns (5'-3'):

[1184] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (e.g., generally a modified unlocked nucleic acid or glycol nucleic acid), and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification.

[1185] Or thus, generally the first strand contains the following modification patterns (5'-3'):

[1186] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, e.g., generally a modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification. Generally, M2 can be the same 2'-sugar modification.

[1187] 17. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand, wherein the second strand comprises:

[1188] Two consecutive abasic nucleosides in the 5' or 3' terminal region of the second strand, and

[1189] Starting from the 5' terminal position 1 of the second strand (position 1 being the nucleoside closest to the 5' end that does not include the abasic nucleoside), position 7 on the second strand comprises a sugar modification (the sugar modification being a 2'-Me modification),

[1190] And optionally wherein the first strand comprises a modification pattern selected from the following or any combination thereof, wherein position 1 is the 5' terminal nucleoside of the first strand and the counting direction is 5'-3':

[1191] 2'-F sugar modifications at least at positions 2, 14, and 16, and / or

[1192] 2'-Me sugar modifications at positions 17 to 23, and / or

[1193] 2'-Me sugar modifications at positions 1, 3 to 5, 10 to 13, and / or

[1194] A 2'-Me sugar modification at position 7 or a heat destabilizing modification at position 7, such as a modified unlocked nucleic acid or glycol nucleic acid typically, and / or

[1195] A 2'-F sugar modification or a heat destabilizing modification at position 6, such as a modified unlocked nucleic acid or glycol nucleic acid typically, and / or

[1196] Positions 8 and 9 can be modifications selected from 2'-Me sugar modifications and 2'-F sugar modifications, and typically can be the same 2'-sugar modification,

[1197] Thus typically the first strand can comprise the following modification pattern (5'-3'):

[1198] Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid typically), which is typically present at position 6, and typically a 2'-Me sugar modification is present at position 7,

[1199] Or thus typically the first strand comprises the following modification pattern (5'-3'):

[1200] Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, such as a commonly modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from 2’-Me sugar modification and 2’-F sugar modification,

[1201] or generally the first strand contains the following modification pattern (5’-3’):

[1202] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a modification selected from the following: 2’-Me sugar modification, 2’-F sugar modification, and heat-destabilizing modification (such as a commonly modified unlocked nucleic acid or glycol nucleic acid), and M2 represents a modification selected from the following: 2’-Me sugar modification and 2’-F sugar modification,

[1203] or generally the first strand contains the following modification pattern (5’-3’):

[1204] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, such as a commonly modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from 2’-Me sugar modification and 2’-F sugar modification, and generally M2 can be the same 2’ sugar modification.

[1205] 18. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region including: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, where the second strand comprises:

[1206] where two phosphorothioate internucleoside bonds are present between three consecutive positions in the 5’ or 3’ terminal region of the second strand, whereby the terminal nucleosides at the 5’ or 3’ terminal region of the second strand are respectively linked to their respective 5’ or 3’ adjacent penultimate nucleosides through phosphorothioate internucleoside bonds, and each 5’ or 3’ penultimate nucleoside is linked to its respective 5’ or 3’ adjacent antepenultimate nucleoside through a phosphorothioate internucleoside bond,

[1207] and

[1208] counting from the 5’ terminal position 1 (excluding the nucleoside closest to the 5’ end without a base) of the second strand, position 7 on the second strand includes a sugar modification (the sugar modification is a 2’-Me modification),

[1209] and optionally wherein the first strand comprises a modification pattern selected from the following or any combination thereof, where position 1 is the 5'-terminal nucleoside of the first strand and the counting direction is 5'-3':

[1210] 2'-F sugar modification at least at positions 2, 14, and 16, and / or

[1211] 2'-Me sugar modification at positions 17 to 23, and / or

[1212] 2'-Me sugar modification at positions 1, 3 to 5, 10 to 13, and / or

[1213] 2'-Me sugar modification at position 7 or a heat destabilizing modification at position 7, such as a modified unlocked nucleic acid or glycol nucleic acid, typically, and / or

[1214] 2'-F sugar modification or a heat destabilizing modification at position 6, such as a modified unlocked nucleic acid or glycol nucleic acid, typically, and / or

[1215] Positions 8 and 9 can be modifications selected from 2'-Me sugar modification and 2'-F sugar modification, and typically can be the same 2'-sugar modification,

[1216] whereby typically the first strand can comprise the following modification pattern (5'-3'):

[1217] Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1218] where M represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and a heat destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid), which is typically present at position 6, and typically a 2'-Me sugar modification is present at position 7, whereby,

[1219] or typically the first strand comprises the following modification pattern (5'-3'):

[1220] Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification,

[1221] or whereby typically the first strand comprises the following modification pattern (5'-3'):

[1222] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a modification selected from the group consisting of: 2'-Me sugar modification, 2'-F sugar modification, and heat destabilizing modification (such as modified unlocked nucleic acid or glycol nucleic acid in general), and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification,

[1223] or thus generally the first strand comprises the following modification pattern (5'-3'):

[1224] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat destabilizing modification, such as modified unlocked nucleic acid or glycol nucleic acid in general, M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification, and generally M2 can be the same 2' sugar modification.

[1225] 19. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand comprises:

[1226] Starting from position 1 at the 5'-end of the second strand (position 1 being the nucleoside closest to the 5'-end that does not include a nucleobase-free nucleoside), position 7 on the second strand comprises a sugar modification (the sugar modification being a 2'-Me modification), and

[1227] At the 3'-end of the second strand, the nucleic acid is directly or indirectly conjugated to one or more ligand moieties, where the ligand moieties generally comprise:

[1228] (i) one or more N-acetylgalactosamine (GalNAc) ligands, and / or

[1229] (ii) one or more derivatives of N-acetylgalactosamine (GalNAc) ligands, and / or

[1230] (iii) one or more N-acetylgalactosamine (GalNAc) ligands and / or their derivatives,

[1231] which are conjugated to the nucleic acid via a linker,

[1232] And optionally, wherein the first strand comprises a modification pattern selected from the following or any combination thereof, where position 1 is the 5'-end nucleoside of the first strand and the counting direction is 5'-3':

[1233] 2'-F sugar modifications at least at positions 2, 14, and 16, and / or

[1234] 2'-Me sugar modifications at positions 17 to 23, and / or

[1235] 2'-Me sugar modifications at positions 1, 3 to 5, 10 to 13, and / or

[1236] a 2'-Me sugar modification at position 7 or a heat-destabilizing modification at position 7, such as a modified unlocked nucleic acid or glycol nucleic acid, typically, and / or

[1237] a 2'-F sugar modification or a heat-destabilizing modification at position 6, such as a modified unlocked nucleic acid or glycol nucleic acid, typically, and / or

[1238] Positions 8 and 9 can be modifications selected from 2'-Me sugar modifications and 2'-F sugar modifications, and typically can be the same 2'-sugar modification,

[1239] Thus, typically, the first strand can contain the following modification pattern (5'-3'):

[1240] Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1241] where M represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid), which is typically present at position 6, and typically a 2'-Me sugar modification is present at position 7,

[1242] Or thus, typically, the first strand contains the following modification pattern (5'-3'):

[1243] Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification,

[1244] Or thus, typically, the first strand contains the following modification pattern (5'-3'):

[1245] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1246] wherein M1 represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (such as a generally modified unlocked nucleic acid or glycol nucleic acid), and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification,

[1247] or thus generally the first strand comprises the following modification pattern (5'-3'):

[1248] Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, such as a generally modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification, and generally M2 can be the same 2'-sugar modification.

[1249] 20. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, the duplex region comprising: a first strand at least partially complementary to a portion of RNA transcribed from the target gene, and a second strand at least partially complementary to the first strand, wherein the second strand comprises the following 2'-sugar and abasic modification pattern:

[1250] Second strand (5'-3'): ia–ia-Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me

[1251] and the first strand comprises a modification pattern (5'-3') selected from the following, wherein position 1 is the 5'-terminal nucleoside of the first strand and the counting direction is 5'-3':

[1252] (5'-3')Me–F–Me–Me–Me–(M)4–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M represents a modification selected from the following: 2'-Me sugar modification, 2'-F sugar modification, and heat-destabilizing modification (such as a generally modified unlocked nucleic acid or glycol nucleic acid), which is generally present at position 6, and generally a 2'-Me sugar modification is present at position 7,

[1253] or thus generally the first strand comprises the following modification pattern (5'-3'):

[1254] (5’-3’)Me–F–Me–Me–Me–(M1)-(M2)3–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid typically, and M2 represents a modification selected from 2’-Me sugar modification and 2’-F sugar modification.

[1255] Or thus typically the first strand comprises the following modification pattern (5’-3’):

[1256] (5’-3’)Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1257] where M1 represents a modification selected from the following: 2’-Me sugar modification, 2’-F sugar modification, and heat-destabilizing modification (such as a modified unlocked nucleic acid or glycol nucleic acid typically), and M2 represents a modification selected from 2’-Me sugar modification and 2’-F sugar modification.

[1258] Or thus typically the first strand comprises the following modification pattern (5’-3’):

[1259] (5’-3’)Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me where M1 represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid typically, M2 represents a modification selected from 2’-Me sugar modification and 2’-F sugar modification, and typically M2 can be the same 2’ sugar modification.

[1260] 21. A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, the duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand and the first strand comprise the following modification patterns:

[1261] Second strand (5’-3’): ia–ia-Me–Me–Me–Me–Me–Me–Me–Me–F–F–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me

[1262] First strand (5’-3’)Me–F–Me–Me–Me–(M1)–Me-(M2)2–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me

[1263] Among them, M1 represents a heat-destabilizing modification, such as a modified unlocked nucleic acid or glycol nucleic acid, and M2 represents a modification selected from 2'-Me sugar modification and 2'-F sugar modification. Usually, M2 can be the same 2'-sugar modification.

[1264] 22. A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand comprises the following 2'-sugar and abasic modification patterns:

[1265] Modification pattern 1:

[1266] Second strand (5'-3'): ia–ia–F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F, optionally combined with the following first strand:

[1267] First strand (5'-3'): Me–F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F–Me,

[1268] Or modification pattern 2:

[1269] Second strand (5'-3'): F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F-ia–ia, optionally combined with the following first strand:

[1270] First strand (5'-3'): Me–F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F–Me

[1271] Wherein:

[1272] ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is usually present in 2-nucleoside overhangs.

[1273] 23. A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the second strand comprises the following 2'-sugar, abasic and bond modification patterns:

[1274] Modification pattern 1:

[1275] Second strand (5’-3’): ia–ia–F(s)Me(s)F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me–F, optionally combined with the following first strand:

[1276] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me(s)F(s)Me,

[1277] Or modification pattern 2:

[1278] Second strand (5’-3’): F–Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me–F(s)Me(s)F-ia–ia, optionally combined with the following first strand:

[1279] First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F-Me-F–Me(s)F(s)Me

[1280] Wherein:

[1281] (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[1282] 24. A nucleic acid for inhibiting the expression of a target gene, which comprises a duplex region, and the duplex region comprises: a first strand that is at least partially complementary to a part of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand, wherein the nucleosides of the second strand and the first strand comprise the following 2’ sugar and bond modification patterns (5’-3’):

[1283] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[1284] Wherein the 2’-Me or 2’-F modified nucleosides of the first strand comprise any one of the following modification patterns (5’-3’):

[1285] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1286] First strand (5’-3’): Me–F–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1287] First strand (5’-3’): Me–F–Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1288] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1289] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1290] First strand (5’-3’): Me–F–Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1291] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me,

[1292] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1293] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1294] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1295] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1296] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1297] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1298] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me,

[1299] where (s) is a phosphorothioate internucleoside linkage.

[1300] 25. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, said duplex region comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand,

[1301] wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar, abasic, and linkage modification patterns (5’-3’):

[1302] Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[1303] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[1304] Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[1305] Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[1306] Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[1307] Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia–ia

[1308] Wherein, the 2’-Me or 2’-F modified nucleosides of the first strand include any one of the following modification patterns (5’-3’):

[1309] First strand (5’-3’): Me–F–Me–Me–Me–F–Me–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1310] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1311] First strand (5’-3’): Me–F–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1312] First strand (5’-3’): Me–F–Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1313] First strand (5’-3’): Me–F–Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1314] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1315] First strand (5’-3’): Me–F–Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me, or

[1316] First strand (5’-3’): Me–F–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–Me–Me,

[1317] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–Me–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1318] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1319] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1320] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1321] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–F–F–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1322] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1323] First strand (5’-3’): Me(s)F(s)Me–Me–Me–F–F–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, or

[1324] First strand (5’-3’): Me(s)F(s)Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me,

[1325] wherein:

[1326] (s) is a phosphorothioate internucleoside bond,

[1327] ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is usually present in two nucleoside overhangs.

[1328] 26. The nucleic acid according to any one of the preceding claims, wherein the first strand comprises at least 17 consecutive nucleosides, the consecutive nucleosides differing from any one of the first strand sequences listed in Table 2 by 0 or 1 nucleoside.

[1329] 27. The nucleic acid according to any one of the preceding claims, wherein the first strand comprises at least 17 consecutive nucleosides, the consecutive nucleosides differing from any one of the first strand sequences listed in Table 3 by 0 or 1 nucleoside.

[1330] 28. The nucleic acid according to claim 26 or 27, wherein the first strand comprises nucleosides 2-18 of any one of the sequences defined in claim 26 or 27.

[1331] 29. The nucleic acid according to any one of the preceding claims, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides, the consecutive nucleosides differing from any one of the second strand sequences listed in Table 2 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand over the 17 consecutive nucleosides.

[1332] 30. The nucleic acid according to any one of the preceding claims, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides, the consecutive nucleosides differing from any one of the second strand sequences listed in Table 4 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand over the 17 consecutive nucleosides.

[1333] 31. The nucleic acid according to any one of the preceding claims, wherein the first strand comprises any one of the first strand sequences listed in Table 2.

[1334] 32. The nucleic acid according to any one of the preceding claims, wherein the first strand comprises any one of the first strand sequences listed in Table 3.

[1335] 33. The nucleic acid according to any one of the preceding claims, wherein the second strand comprises any one of the second strand sequences listed in Table 2.

[1336] 34. A nucleic acid according to any one of the foregoing, wherein the second strand comprises any one of the second strand sequences listed in Table 4.

[1337] 35. A nucleic acid according to any one of the foregoing, wherein the nucleic acid is an siRNA oligonucleotide.

[1338] 36. A nucleic acid according to any one of the foregoing, wherein the nucleic acid is directly or indirectly coupled to one or more ligand moieties, optionally wherein the ligand moiety is present in the terminal region of the second strand, typically in its 3' terminal region.

[1339] 37. A nucleic acid according to item 36, wherein the ligand moiety comprises:

[1340] (i) one or more N-acetylgalactosamine (GalNAc) ligands, and / or

[1341] (ii) one or more derivatives of N-acetylgalactosamine (GalNAc) ligands, and / or

[1342] (iii) one or more N-acetylgalactosamine (GalNAc) ligands and / or their derivatives,

[1343] which is coupled to the nucleic acid through a linker.

[1344] 38. A nucleic acid according to item 37, wherein the one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly coupled to the 5' or 3' terminal region of the second strand of the nucleic acid, typically coupled to its 3' terminal region.

[1345] 39. A nucleic acid according to any one of items 36 to 38, wherein the ligand moiety comprises the following structure:

[1346]

[1347] 40. A nucleic acid according to any one of items 36 to 39, having the following structure:

[1348]

[1349] wherein:

[1350] R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl each time it appears;

[1351] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro;

[1352] X1 and X2 are independently selected from the following each time they appear: methylene, oxygen, and sulfur;

[1353] m is an integer from 1 to 6;

[1354] n is an integer from 1 to 10;

[1355] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1356] (i) q and r cannot both be 0; and

[1357] (ii) s, t, and v cannot all be 0;

[1358] Z is an oligonucleoside.

[1359] 41. The nucleic acid according to any one of items 36 to 39 has the following structure:

[1360]

[1361] wherein: r and s are independently integers selected from 1 to 16; and

[1362] Z is an oligonucleoside.

[1363] 42. A pharmaceutical composition comprising the nucleic acid according to any one of the foregoing, and a pharmaceutically acceptable excipient or carrier.

[1364] 43. Use of the nucleic acid or pharmaceutical composition according to any one of the foregoing for treatment.

[1365] 44. Use of the nucleic acid or pharmaceutical composition according to any one of the foregoing for preventing or treating a disease associated with a hemostatic disorder, such as a disease associated with a hemostatic disorder (such as hemophilia).

[1366] 45. Use of the nucleic acid or pharmaceutical composition according to any one of the foregoing for preventing or treating diabetes.

[1367] 46. Use of the nucleic acid or pharmaceutical composition according to any one of the foregoing for preventing or treating cardiovascular diseases.

[1368] In one aspect, the present application can be applied to the following compounds, methods, compositions or uses numbered 1 - 101, wherein any reference to a formula in items 1 - 101 refers only to those formulas defined within items 1 - 101. These formulas are reproduced in Figure 5 Specifically, the oligonucleoside moiety represented by Z in any of the following may comprise a nucleic acid for inhibiting the expression of ZPI or HCII as defined below.

[1369] 1. A compound comprising the following structure:

[1370]

[1371] wherein:

[1372] Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl;

[1373] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro;

[1374] Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen, and sulfur;

[1375] m is an integer from 1 to 6;

[1376] n is an integer from 1 to 10;

[1377] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1378] (i) q and r cannot both be 0; and

[1379] (ii) s, t, and v cannot all be 0;

[1380] Z is an oligonucleotide moiety.

[1381] 2. The compound according to item 1, wherein each occurrence of R1 is hydrogen.

[1382] 3. The compound according to item 1, wherein R1 is methyl.

[1383] 4. The compound according to item 1, wherein R1 is ethyl.

[1384] 5. The compound according to any one of items 1 to 4, wherein R2 is hydroxy.

[1385] 6. The compound according to any one of items 1 to 4, wherein R2 is halogen.

[1386] 7. The compound according to item 6, wherein R2 is fluorine.

[1387] 8. The compound according to item 6, wherein R2 is chlorine.

[1388] 9. The compound according to item 6, wherein R2 is bromine.

[1389] 10. The compound according to item 6, wherein R2 is iodine.

[1390] 11. The compound according to item 6, wherein R2 is nitro.

[1391] 12. The compound according to any one of items 1 to 11, wherein X1 is methylene.

[1392] 13. The compound according to any one of items 1 to 11, wherein X1 is oxygen.

[1393] 14. The compound according to any one of items 1 to 11, wherein X1 is sulfur.

[1394] 15. The compound according to any one of items 1 to 14, wherein X2 is methylene.

[1395] 16. The compound according to any one of items 1 to 15, wherein X2 is oxygen.

[1396] 17. The compound according to any one of items 1 to 16, wherein X2 is sulfur.

[1397] 18. The compound according to any one of items 1 to 17, wherein m = 3.

[1398] 19. The compound according to any one of items 1 to 18, wherein n = 6.

[1399] 20. The compound according to items 13 and 15, wherein X1 is oxygen and X2 is methylene, and preferably wherein:

[1400] q = 1,

[1401] r = 2,

[1402] s = 1,

[1403] t = 1,

[1404] v = 1.

[1405] 21. The compound according to items 12 and 15, wherein both X1 and X2 are methylene, and preferably wherein:

[1406] q = 1,

[1407] r = 3,

[1408] s = 1,

[1409] t = 1,

[1410] v = 1.

[1411] 22. The compound according to any one of items 1 to 21, wherein Z is:

[1412]

[1413] wherein:

[1414] Z1, Z2, Z3, Z4 are each independently oxygen or sulfur upon each occurrence; and one of the bonds between P and Z2 and between P and Z3 is a single bond and the other is a double bond.

[1415] 23. The compound according to item 22, wherein the oligonucleoside is an RNA compound capable of regulating, preferably inhibiting, the expression of a target gene.

[1416] 24. The compound according to item 23, wherein the RNA compound comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5'-end and a 3'-end.

[1417] 25. The compound according to item 24, wherein the RNA compound is linked to an adjacent phosphate group at the 5'-end of its second strand.

[1418] 26. The compound according to item 24, wherein the RNA compound is linked to an adjacent phosphate group at the 3'-end of its second strand.

[1419] 27. A compound of formula (II):

[1420]

[1421] 28. A compound of formula (III):

[1422]

[1423] 29. The compound according to item 27 or 28, wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5'-end and a 3'-end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5'-end of its second strand.

[1424] 30. A composition comprising a compound of formula (II) as defined in item 27, and a compound of formula (III) as defined in item 28, optionally subject to item 29.

[1425] 31. The composition according to item 30, wherein the compound of formula (III) as defined in item 28 is present in an amount of 10 to 15 wt% of the composition.

[1426] 32. A compound of formula (IV)

[1427]

[1428] 33. A compound of formula (V):

[1429]

[1430] 34. A compound according to item 32 or 33, wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1431] 35. A composition comprising a compound of formula (IV) as defined in item 32, and a compound of formula (V) as defined in item 33, optionally subordinate to item 34.

[1432] 36. The composition according to item 35, wherein the compound of formula (V) as defined in item 33 is present in an amount of 10 to 15 wt% of the composition.

[1433] 37. A compound as defined in any one of items 1 to 29 or items 32 to 34, wherein the oligonucleoside comprises an RNA duplex which further comprises one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[1434] 38. The compound according to item 37, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[1435] 39. The compound according to any one of items 1 to 29, or 32 to 34, or 37 to 38, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more ends.

[1436] 40. The compound according to item 39, wherein the one or more degradation protection moieties are not at the ends of the oligonucleoside chain carrying the ligand moiety, and / or wherein the one or more degradation protection moieties are selected from the following: phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein the inverted abasic nucleosides are present at the distal end of the chain carrying the ligand moiety.

[1437] 41. The compound according to any one of items 1 to 29, or 32 to 34, or 37 to 40, wherein the ligand moiety shown in formula (I) in item 1 comprises one or more ligands.

[1438] 42. The compound according to item 41, wherein the ligand moiety shown in formula (I) in item 1 comprises one or more carbohydrate ligands.

[1439] 43. The compound according to item 42, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides or polysaccharides.

[1440] 44. The compound according to item 43, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[1441] 45. The compound according to item 44, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[1442] 46. The compound according to item 45, which comprises two or three N-acetylgalactosamine moieties.

[1443] 47. The compound according to any one of items 41 to 46, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[1444] 48. The compound according to item 47, wherein the one or more ligands are linked in a bi-antennary or tri-antennary branched configuration.

[1445] 49. The compound according to items 46 to 48, wherein the moiety shown in formula (I) in item 1:

[1446]

[1447] is any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa)

[1448]

[1449] wherein:

[1450] A I is hydrogen, or a suitable hydroxyl protecting group;

[1451] a is an integer of 2 or 3; and

[1452] b is an integer from 2 to 5; or

[1453] wherein:

[1454] A I is hydrogen, or a suitable hydroxyl protecting group;

[1455] a is an integer of 2 or 3; and

[1456] c and d are independently integers from 1 to 6; or

[1457] wherein:

[1458] A I is hydrogen, or a suitable hydroxyl protecting group;

[1459] a is an integer of 2 or 3; and

[1460] e is an integer from 2 to 10.

[1461] 50. The compound according to items 46 to 48, wherein the moiety shown in formula (I) in item 1:

[1462]

[1463] wherein:

[1464] A I is hydrogen;

[1465] a is an integer of 2 or 3.

[1466] 51. The compound according to item 49 or 50, wherein a = 2.

[1467] 52. The compound according to item 49 or 50, wherein a = 3.

[1468] 53. The compound according to item 49, wherein b = 3.

[1469] 54. The compound of formula (VIII):

[1470]

[1471] 55. The compound of formula (VIII):

[1472]

[1473]

[1474] 56. The compound according to item 54 or 55, wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1475] 57. A composition comprising the compound of formula (VIII) as defined in item 54, and the compound of formula (IX) as defined in item 55, optionally subject to item 56.

[1476] 58. The composition according to item 57, wherein the compound of formula (IX) as defined in item 55 is present in an amount of 10 to 15 wt% of the composition.

[1477] 59. The compound of formula (X):

[1478]

[1479] 60. Compound of formula (XI):

[1480]

[1481] 61. The compound according to item 59 or 60, wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5'-end and a 3'-end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3'-end of its second strand.

[1482] 62. A composition comprising the compound of formula (X) as defined in item 59, and the compound of formula (XI) as defined in item 60, optionally subject to item 61.

[1483] 63. The composition according to item 62, wherein the compound of formula (XI) as defined in item 60 is present in an amount of 10 to 15 wt% of the composition.

[1484] 64. The compound as defined in any one of items 54 to 63, wherein the oligonucleoside comprises an RNA duplex which further comprises one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[1485] 65. The compound according to item 64, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[1486] 66. The compound according to any one of items 54 to 65, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more ends.

[1487] 67. The compound according to item 66, wherein the one or more degradation protection moieties are not at the ends of the oligonucleoside chain carrying the ligand moiety, and / or wherein the one or more degradation protection moieties are selected from the following: phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages and inverted abasic nucleosides, wherein the inverted abasic nucleoside is present at the distal end of the chain carrying the ligand moiety, as shown in any one of formula (VIII), (IX), (X) or (XI) in any one of items 54, 55, 59 or 60.

[1488] 68. A method for preparing the compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67 and / or the composition according to any one of items 30, 31, 35, 36, 57, 58, 62, 63, which comprises reacting compounds of formula (XII) and (XIII):

[1489]

[1490] Wherein:

[1491] Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl;

[1492] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro;

[1493] Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen, and sulfur;

[1494] m is an integer from 1 to 6;

[1495] n is an integer from 1 to 10;

[1496] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1497] (i) q and r cannot both be 0; and

[1498] (ii) s, t, and v cannot all be 0;

[1499] Z is an oligonucleoside moiety;

[1500] And, where appropriate, deprotection of the ligand and / or annealing of the second strand of the oligonucleoside moiety is carried out.

[1501] 69. The method according to item 68, wherein the compound of formula (XII) is prepared by reacting the compounds of formula (XIV) and (XV):

[1502]

[1503] Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl;

[1504] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro;

[1505] Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen, and sulfur;

[1506] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1507] (i) q and r cannot both be 0; and

[1508] (ii) s, t, and v cannot all be 0;

[1509] Z is an oligonucleotide moiety.

[1510] 70. The method according to item 68, for preparing a compound according to any one of items 20, 25, 27, 29, 54, 56, and / or a composition according to any one of items 30, 31, 57, 58, wherein: the compound of formula (XII) is of formula (XIIa):

[1511]

[1512]

[1513] and the compound of formula (XIII) is of formula (XIIIa):

[1514]

[1515] wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5'-end and a 3'-end, and wherein the RNA duplex is linked at the 5'-end of its second strand to an adjacent phosphate group.

[1516] 71. The method according to item 68, for preparing a compound according to any one of items 20, 25, 28, 29, 55, 56, and / or a composition according to any one of items 30, 31, 57, 58, wherein:

[1517] the compound of formula (XII) is of formula (XIIb):

[1518]

[1519] and the compound of formula (XIII) is of formula (XIIIa):

[1520]

[1521] wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5'-end and a 3'-end, and wherein the RNA duplex is linked at the 5'-end of its second strand to an adjacent phosphate group.

[1522] 72. The method according to item 68, for preparing a compound according to any one of items 21, 26, 32, 34, 59, 61, and / or a composition according to any one of items 35, 36, 62, 63, wherein:

[1523] The compound of formula (XII) is of formula (XIIc):

[1524]

[1525] And the compound of formula (XIII) is of formula (XIIIa):

[1526]

[1527] Wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1528] 73. The method according to item 68, for preparing the compound according to any one of items 21, 26, 33, 34, 60, 61, and / or the composition according to any one of items 35, 36, 62, 63, wherein:

[1529] The compound of formula (XII) is of formula (XIId):

[1530]

[1531] And the compound of formula (XIII) is of formula (XIIIa):

[1532]

[1533] Wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1534] 74. The method according to any one of items 70 to 73, wherein:

[1535] The compound of formula (XIIIa) is of formula (XIIIb):

[1536]

[1537] 75. The method according to item 69, subordinate to items 70 to 73, wherein:

[1538] The compound of formula (XIV) is of formula (XIVa) or formula (XIVb):

[1539]

[1540] and the compound of formula (XV) is of formula (XVa) or formula (XIVb):

[1541]

[1542]

[1543] wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5'-end and a 3'-end, and wherein (i) the RNA duplex is linked at the 5'-end of its second strand to an adjacent phosphate group in formula (XVa), or (ii) the RNA duplex is linked at the 3'-end of its second strand to an adjacent phosphate group in formula (XVb).

[1544] 76. The compound of formula (XII):

[1545]

[1546] wherein:

[1547] R1 is independently selected from the group consisting of hydrogen, methyl and ethyl each time it appears;

[1548] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro;

[1549] X1 and X2 are independently selected from the group consisting of methylene, oxygen and sulfur each time they appear;

[1550] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1551] (i) q and r cannot both be 0; and

[1552] (ii) s, t and v cannot all be 0;

[1553] Z is an oligonucleoside moiety.

[1554] 77. The compound of formula (XIIa):

[1555]

[1556] 78. The compound of formula (XIIb):

[1557]

[1558] 79. The compound of formula (XIIc):

[1559]

[1560] 80. Compounds of formula (XIId):

[1561]

[1562] 81. Compounds of formula (XIII):

[1563]

[1564] in:

[1565] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1566] m is an integer from 1 to 6;

[1567] n is an integer of 1 to 10.

[1568] 82. Compounds of formula (XIIIa):

[1569]

[1570] 83. Compounds of formula (XIIIb):

[1571]

[1572] 84. Compounds of formula (XIV):

[1573]

[1574] in:

[1575] R1 is selected from the group consisting of hydrogen, methyl and ethyl;

[1576] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[1577] X2 is selected from the group consisting of methylene, oxygen and sulfur;

[1578] s, t, and v are independently integers from 0 to 4, provided that s, t, and v cannot be 0 at the same time.

[1579] 85. Compounds of formula (XIVa):

[1580]

[1581] 86. Compounds of formula (XIVb):

[1582]

[1583] 87. Compound of formula (XV):

[1584]

[1585] Wherein:

[1586] Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl;

[1587] X1 is selected from the group consisting of methylene, oxygen, and sulfur;

[1588] q and r are independently integers from 0 to 4, provided that q and r cannot both be 0;

[1589] Z is an oligonucleoside moiety.

[1590] 88. Compound of formula (XVa):

[1591]

[1592] 89. Compound of formula (XVb):

[1593]

[1594] 90. Use of the compound according to any one of items 76, 81 to 84, 87 in the preparation of the compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67 and / or the composition according to any one of items 30, 31, 35, 36, 57, 58, 62, and 63.

[1595] 91. Use of the compound according to item 85 in the preparation of the compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or the composition according to any one of items 30, 31, 35, 36, 57, 58, 62, and 63, wherein R2 = F.

[1596] 92. Use of the compound according to item 86 in the preparation of the compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or the composition according to any one of items 30, 31, 35, 36, 57, 58, 62, and 63, wherein R2 = OH.

[1597] 93. Use of the compound according to item 77 in the preparation of the compound according to any one of items 20, 25, 27, 29, 54, 56 and / or the composition according to any one of items 30, 31, 57, 58.

[1598] Use of the compound according to item 78 in the preparation of the compound according to any one of items 20, 25, 28, 29, 55, 56 and / or the composition according to any one of items 30, 31, 57, 58.

[1599] 95. Use of the compound according to item 79 in the preparation of the compound according to any one of items 21, 26, 32, 34, 59, 61 and / or the composition according to any one of items 35, 36, 62, 63.

[1600] 96. Use of the compound according to item 80 in the preparation of the compound according to any one of items 21, 26, 33, 34, 60, 61 and / or the composition according to any one of items 35, 36, 62, 63.

[1601] 97. Use of the compound according to item 88 in the preparation of the compound according to any one of items 20, 25, 27 to 29, 54 to 56 and / or the composition according to any one of items 30, 31, 57, 58.

[1602] 98. Use of the compound according to item 89 in the preparation of the compound according to any one of items 21, 26, 32 to 34, 59 to 61 and / or the composition according to any one of items 35, 36, 62, 63.

[1603] 99. A compound or composition obtained or obtainable by the method according to any one of items 68 to 75.

[1604] 100. A pharmaceutical composition comprising the compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or the composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63, and a pharmaceutically acceptable carrier, diluent or excipient.

[1605] 101. The compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or the composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63, for treatment.

[1606] In another aspect, the present application can be applied to the following compounds, methods, compositions or uses numbered as Clauses 1 - 56, wherein any reference to a formula in the clauses refers only to those formulas defined within Clauses 1 - 56. These formulas are reproduced in Figure 6 Specifically, the oligonucleotide moiety represented by Z in any of the following clauses may comprise a nucleic acid for inhibiting the expression of ZPI or HCII defined below.

[1607] 1. A compound comprising the following structure:

[1608]

[1609] Wherein:

[1610] r and s are independently integers selected from 1 to 16; and

[1611] Z is an oligonucleotide moiety.

[1612] 2. The compound according to clause 1, wherein s is an integer selected from 4 to 12.

[1613] 3. The compound according to clause 2, wherein s is 6.

[1614] 4. The compound according to any one of clauses 1 to 3, wherein r is an integer selected from 4 to 14.

[1615] 5. The compound according to clause 4, wherein r is 6.

[1616] 6. The compound according to clause 4, wherein r is 12.

[1617] 7. The compound according to clause 5, which is subordinate to clause 3.

[1618] 8. The compound according to clause 6, which is subordinate to clause 3.

[1619] 9. The compound according to any one of clauses 1 to 8, wherein Z is:

[1620]

[1621] Wherein:

[1622] Z1, Z2, Z3, Z4 are independently oxygen or sulfur each time they appear; and

[1623] One bond between P and Z2 and the bond between P and Z3 is a single bond and the other is a double bond.

[1624] 10. The compound according to any one of clauses 1 to 9, wherein the oligonucleotide is an RNA compound capable of regulating, preferably inhibiting, the expression of a target gene.

[1625] 11. The compound according to clause 10, wherein the RNA compound comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end.

[1626] 12. The compound according to clause 11, preferably also subordinate to clauses 3 and 6, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[1627] 13. The compound according to clause 11, preferably also subordinate to clauses 3 and 5, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[1628] 14. The compound of formula (II), preferably subordinate to clause 12:

[1629]

[1630] 15. The compound of formula (III), preferably subordinate to clause 13:

[1631]

[1632] 16. The compound as defined in any one of clauses 1 to 15, wherein the oligonucleoside comprises an RNA duplex, and the RNA duplex further comprises one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[1633] 17. The compound according to clause 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[1634] 18. The compound according to any one of clauses 1 to 17, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more ends.

[1635] 19. The compound according to clause 18, wherein the one or more degradation protection moieties are not present at the ends of the oligonucleoside chain carrying the linker / ligand moiety, and / or wherein the one or more degradation protection moieties are selected from the following: phosphorothioate internucleoside bonds, phosphorodithioate internucleoside bonds, and inverted abasic nucleosides, wherein the inverted abasic nucleosides are present at the distal end of the same strand as the end carrying the linker / ligand moiety.

[1636] 20. The compound according to any one of clauses 1 to 19, wherein the ligand moiety shown in formula (I) in clause 1 comprises one or more ligands.

[1637] 21. The compound according to clause 20, wherein the ligand moiety shown in formula (I) in clause 1 comprises one or more carbohydrate ligands.

[1638] 22. The compound according to clause 21, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[1639] 23. The compound according to clause 22, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[1640] 24. The compound according to clause 23, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[1641] 25. The compound according to clause 24, which comprises two or three N-acetylgalactosamine moieties.

[1642] 26. The compound according to any one of the preceding clauses, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[1643] 27. The compound according to clause 26, wherein the one or more ligands are linked in a biantennary or triantennary branched configuration.

[1644] 28. The compound according to clauses 20 to 27, wherein the moiety shown in formula (I) in clause 1:

[1645]

[1646] is any one of formula (IV), (V) or (VI), preferably formula (IV):

[1647]

[1648] wherein:

[1649] A1 is hydrogen, or a suitable hydroxyl protecting group;

[1650] a is an integer of 2 or 3; and

[1651] b is an integer from 2 to 5; or

[1652]

[1653] wherein:

[1654] A1 is hydrogen, or a suitable hydroxyl protecting group;

[1655] a is an integer of 2 or 3; and

[1656] c and d are independently integers from 1 to 6; or

[1657]

[1658] wherein:

[1659] A1 is hydrogen, or a suitable hydroxyl protecting group;

[1660] a is an integer of 2 or 3; and

[1661] e is an integer from 2 to 10.

[1662] 29. The compound according to any one of Clauses 1 to 28, wherein the moiety shown in Formula (I) of Clause 1:

[1663]

[1664] wherein:

[1665] A1 is hydrogen;

[1666] a is an integer of 2 or 3.

[1667] 30. The compound according to Clause 28 or 29, wherein a = 2.

[1668] 31. The compound according to Clause 28 or 29, wherein a = 3.

[1669] 32. The compound according to Clause 28, wherein b = 3.

[1670] 33. The compound of Formula (VIII):

[1671]

[1672] 34. The compound of Formula (IX):

[1673]

[1674] 35. The compound according to Clause 33 or 34, wherein the oligonucleoside comprises an RNA duplex, and the RNA duplex further comprises one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[1675] 36. The compound according to Clause 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[1676] 37. The compound according to any one of Clauses 33 to 36, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more ends.

[1677] 38. The compound according to Clause 37, wherein the one or more degradation protection moieties are not present at the ends of the oligonucleoside chain carrying the linker / ligand moiety, and / or wherein the one or more degradation protection moieties are selected from the following: phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein the inverted abasic nucleosides are present at the distal end of the same chain as the end carrying the linker / ligand moiety.

[1678] 39. The compound according to clause 33, wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1679] 40. The compound according to clause 34, wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1680] 41. A method for preparing the compound according to any one of clauses 1 to 40, which comprises reacting compounds of formula (X) and (XI):

[1681]

[1682] wherein: r and s are independently integers selected from 1 to 16; and

[1683] Z is an oligonucleoside moiety; and deprotection of the ligand and / or annealing of the second strand of the oligonucleoside are carried out as appropriate.

[1684] 42. The method according to clause 41, for preparing the compound according to any one of clauses 6, 8 to 14, 16 to 33, and 35 to 40,

[1685] wherein: the compound of formula (X) is of formula (Xa):

[1686]

[1687] Formula (Xa)

[1688] and the compound of formula (XI) is of formula (XIa):

[1689]

[1690] wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1691] 43. The method according to clause 41, for preparing a compound according to any one of clauses 5, 7, 9 to 13, 15 to 32 and 34 to 40, wherein:

[1692] The compound of formula (X) is of formula (Xb):

[1693]

[1694] and the compound of formula (XI) is of formula (XIa):

[1695]

[1696] wherein the oligonucleoside comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked at the 3' end of its second strand to an adjacent phosphate group.

[1697] 44. The method according to clause 42 or 43, wherein:

[1698] The compound of formula (XIa) is of formula (XIb):

[1699]

[1700] 45. A compound of formula (X):

[1701]

[1702] wherein:

[1703] r is independently an integer selected from 1 to 16; and

[1704] Z is an oligonucleoside moiety.

[1705] 46. A compound of formula (Xa):

[1706]

[1707] Formula (Xa)

[1708] 47. A compound of formula (Xb):

[1709]

[1710] 48. A compound of formula (XI):

[1711]

[1712] wherein:

[1713] s is independently an integer selected from 1 to 16; and

[1714] Z is an oligonucleotide moiety.

[1715] 49. A compound of formula (XIa):

[1716]

[1717] 50. A compound of formula (XIb):

[1718]

[1719] 51. Use of a compound as described in any one of clauses 45 and 48 to 50 in the preparation of a compound as described in any one of clauses 1 to 40.

[1720] 52. Use of a compound as described in clause 46 in the preparation of a compound as described in any one of clauses 6, 8 to 14, 16 to 33 and 35 to 40.

[1721] 53. Use of a compound as described in clause 47 in the preparation of a compound as described in any one of clauses 5, 7, 9 to 13, 15 to 32 and 34 to 40.

[1722] 54. A compound or composition obtainable or obtained by a method as described in any one of clauses 41 to 44.

[1723] 55. A pharmaceutical composition comprising a compound as described in any one of clauses 1 to 40 and a pharmaceutically acceptable carrier, diluent or excipient.

[1724] 56. A compound as described in any one of clauses 1 to 40 for use in therapy.

[1725] Examples

[1726] The present application will be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the present application. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes can be made by those skilled in the art, and such modifications or changes should be included within the spirit and scope of the present application and the scope of the additional claims.

[1727] Example 1: Synthesis of Linker 1

[1728] General experimental conditions:

[1729] Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using Macherey-Nagel's 254 nm fluorescent indicator. Compounds were visualized under ultraviolet light (254 nm) or by spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to the method of Stahl (from Sigma-Aldrich) and then heating. Flash chromatography was carried out using a Biotage Isolera One flash chromatography instrument equipped with a dual-variable ultraviolet wavelength detector (200 - 400 nm) and using Biotage 10, 25, 50 or 100 g chromatography columns (Uppsala, Sweden).

[1730] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH+Co.KG. D-galactosamine pentaacetate was purchased from AK scientific.

[1731] HPLC / ESI-MS was performed using a Waters Acquity UPLC Protein BEH C4 column( 1.7 μm, 2.1x100 mm) at 60 °C on a Dionex UltiMate 3000RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A (H2O containing 0.1% formic acid) and solvent B (acetonitrile (ACN) containing 0.1% formic acid). A flow rate of 0.4 mL / min was used, and the gradient of B increased from 5% to 100% within 15 min. Detector and conditions: Corona charged aerosol detector (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[1732] 1H NMR) and 125 MHz ( 1 13C NMR) were recorded at room temperature on a Varian spectrometer 13 at 500 MHz( 1 1H and 13 13C NMR. Chemical shifts were in ppm, referenced to the solvent residual peaks (CDCl3 – 1 1H NMR: δ at 7.26 ppm, 13 13C NMR δ at 77.2 ppm; DMSO-d6 – 1H NMR: δ at 2.50 ppm, 1313C NMR δ (at 39.5 ppm). Coupling constants are in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[1733] Synthetic route of the coupling building block TriGalNAc - tether 1:

[1734]

[1735] Preparation of compound 2: Under argon, D - galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (DCM) (30 mL), and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq) was added. The reaction was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aqueous NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4 and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0 - 10% MeOH in DCM, 10 CV). A colorless oil product was obtained (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1736]

[1737] Preparation of compound 4: Under argon, compound 2 (2.30 g, 6.98 mmol, 1.0 eq) and azido - PEG3 - OH (1.83 g, 10.5 mmol, 1.5 eq) were dissolved in anhydrous DCM (40 mL), and molecular sieves (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. Then TMSOTf (0.77 g, 3.49 mmol, 0.5 eq) was added to the mixture and the reaction was stirred overnight. The molecular sieves were filtered off, and the filtrate was diluted with DCM (100 mL) and washed with cold saturated aqueous NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4 and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0 - 3% MeOH in DCM, 10 CV) to give the title compound as a pale yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: Calculated for C 20 H 32 N4O 11 , 504.21. Found 505.4. 11H NMR (500 MHz, CDCl3) δ 6.21 - 6.14 (m, 1H), 5.30 (dd, J = 3.4, 1.1 Hz, 1H), 5.04 (dd, J = 11.2, 3.4 Hz, 1H), 4.76 (d, J = 8.6 Hz, 1H), 4.23 - 4.08 (m, 3H), 3.91 - 3.80 (m, 3H), 3.74 - 3.59 (m, 9H), 3.49 - 3.41 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.97 (d, J = 4.2 Hz, 6H). 13 13C NMR (125 MHz, CDCl3) δ 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).

[1738]

[1739] Preparation of Compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 equiv) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1:1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed by vacuum / argon cycling (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through Celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to give the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: Calculated for C 20 H 34 N2O 11 , 478.2. Found 479.4.

[1740]

[1741] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 equiv) was dissolved in a mixture of DCM / water (40 mL 1:1 v / v), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 equiv) was added while stirring vigorously. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 equiv) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% EtOAc in cyclohexane, 12 CV) to give the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: calculated for C 33 H 53 NO 11 , 639.3. Found 640.9. 1 H NMR (500 MHz, DMSO-d6) δ 7.38 - 7.26 (m, 5H), 4.97 (s, 2H), 3.54 (t, 6H), 3.50 (s, 6H), 2.38 (t, 6H), 1.39 (s, 27H). 13 C NMR (125 MHz, DMSO-d6) δ 170.3 (3xC), 154.5 (C), 137.1 (C), 128.2 (2xCH), 127.7 (CH), 127.6 (2xCH), 79.7 (3xC), 68.4 (3xCH2), 66.8 (3xCH2), 64.9 (C), 58.7 (CH2), 35.8 (3xCH2), 27.7 (9xCH3).

[1742]

[1743] Preparation of Compound 8: Under argon, Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 equiv) was dissolved in CH2Cl2 (1 mL), trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to give the TFA salt of the compound (0.183 g, 98%). This compound was used without further purification. MS: calculated for C 21 H 29 NO 11 , 471.6. Found 472.4.

[1744]

[1745] Preparation of Compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 equiv) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 equiv) were dissolved in N,N-dimethylformamide (DMF) (25 mL). Then N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 equiv), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 equiv) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 equiv) were added to the solution, and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and washed with saturated aqueous NaHCO3 solution (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude product was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 14 CV). The product was obtained as a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: calculated for C 81 H 125 N7O 41 , 1852.9. Found 1854.7. 1 H NMR (500 MHz, DMSO-d6) δ 7.90 - 7.80 (m, 10H), 7.65 - 7.62 (m, 4H), 7.47 - 7.43 (m, 3H), 7.38 - 7.32 (m, 8H), 5.24 - 5.22 (m, 3H), 5.02 - 4.97 (m, 4H), 4.60 - 4.57 (m, 3H), 4.07 - 3.90 (m 10H), 3.67 - 3.36 (m, 70H), 3.23 - 3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80 - 1.78 (m, 17H). 1313C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[1746]

[1747] Preparation of Compound 10: Dissolve the trifunctional GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 equiv) in MeOH (15 mL), add 3 drops of acetic acid (AcOH) and Pd / C (30 mg). The reaction mixture was degassed by vacuum / argon cycling (3x) and hydrogenated overnight under balloon pressure. The completion of the reaction was monitored by mass spectrometry, and the resulting mixture was filtered through a thin pad of diatomaceous earth. The solvent was evaporated and the obtained residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellow oil (0.24 g, quantitative yield). MS: Calculated for C 73 H 119 N7O 39 , 1718.8. Found 1719.3.

[1748]

[1749] Preparation of Compound 11: Dissolve commercially available suberic acid bis(N-hydroxysuccinimide ester) (3.67 g, 9.9 mmol, 1.0 equiv) in DMF (5 mL), and add triethylamine (1.2 mL). To this solution was added dropwise a solution of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0) in DMF (5 mL). The reaction was stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4 and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 16 CV). The product was obtained as a white solid (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM)). MS: Calculated for C 15 H 23 N5O5, 353.4.

[1750] The actual value is 354.3.

[1751]

[1752] Preparation of TriGalNAc(12): Under argon, the trisialyl GalNAc compound 10 (0.35 g, 0.24 mmol, 1.0 equiv) and compound 11 (0.11 g, 0.31 mmol, 1.5 equiv) were dissolved in DCM (5 mL), and triethylamine (0.1 mL, 0.61 mmol, 3.0 equiv) was added. The reaction was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was evaporated, and the resulting crude material was purified by flash chromatography (elution gradient: 0 - 10% MeOH in DCM, 20 CV) to give the title compound as a white fluffy solid (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)). MS: Calculated for C 84 H 137 N 11 O 41 , 1957.1. The actual value is 1959.6.

[1753] Coupling of the linker 1 with the siRNA strand: Monofluoro cyclooctyne (MFCO) coupling at the 5’ or 3’ end

[1754] 5’-end MFCO coupling

[1755]

[1756] 3’-end MFCO coupling

[1757]

[1758] General conditions for MFCO coupling: The amine-modified single strand was dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer (pH 9.6) / dimethyl sulfoxide (DMSO) (4:6 (v / v)), and a solution of one molar equivalent of 35 mM MFCO-C6-NHS ester (Berry & Associates, catalog number: #LK 4300) in DMF was added to this solution. The reaction was carried out at room temperature, and after 1 h, another molar equivalent of the MFCO solution was added. The reaction was carried out for another hour and monitored by LC / MS. At least two molar equivalents excess of the MFCO NHS ester reagent relative to the amino-modified oligonucleotide was required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered through a 1.2 μm filter from Sartorius, and then at Purification was carried out by reverse phase (RP HPLC) on a Pure instrument (GE Healthcare).

[1759] Purification was carried out using a Waters XBridge C18 Prep 19x50 mm column. Buffer A was 100 mM TEAAc pH 7 and buffer B contained 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60 °C. UV traces were recorded at 280 nm. A gradient of 0–100% B in 60 column volumes was used.

[1760] The fractions containing the full-length conjugated oligonucleotides were pooled, precipitated in the refrigerator with 3 M NaOAc, pH 5.2 and 85% ethanol, and the collected precipitate was dissolved in water. The sample was desalted by size exclusion chromatography and concentrated using a speed-vac concentrator to give the conjugated oligonucleotides with a separation yield of 40–80%.

[1761] 5’-GalNAc-T1 conjugate

[1762]

[1763] 3’-GalNAc-T1 conjugate

[1764]

[1765] General procedure for conjugation: The MFCO-modified single strand was dissolved in water at 2000 OD / mL and a solution of one equivalent of compound 12 (10 mM) in DMF was added to this solution. The reaction was carried out at room temperature and after 3 h a solution of 0.7 molar equivalents of compound 12 was added. The reaction was carried out overnight and the completion was monitored by LCMS. The conjugate was diluted 15-fold in water, filtered through a 1.2 μm filter from Sartorius, and then purified by RP HPLC on a Pure instrument (GE Healthcare).

[1766] RP HPLC purification was carried out using a Waters XBridge C18 Prep 19x50 mm column. Buffer A was 100 mM triethylammonium acetate (pH 7) and buffer B contained 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60 °C. UV traces were recorded at 280 nm. A gradient of 0–100% B in 60 column volumes was used.

[1767] Fractions containing the fully-length conjugated oligonucleotides were pooled, precipitated in the refrigerator with 3M NaOAc, pH 5.2 and 85% ethanol, and the collected precipitate was dissolved in water to obtain an oligonucleotide solution of approximately 1000 OD / mL. O-acetates were removed by adding 20% ammonia water. Quantitative removal of these protecting groups was verified by LC-MS.

[1768] Desalting of the conjugate was performed by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on a Pure (GE Healthcare) instrument to obtain the conjugated oligonucleotide with a separation yield of 50 - 70%.

[1769] The following protocol further lists the synthetic route:

[1770] Scheme 1:

[1771]

[1772] Scheme 2:

[1773]

[1774] Scheme 3:

[1775]

[1776] Scheme 4:

[1777]

[1778] Scheme 5:

[1779]

[1780] Example 2: Double Annealing

[1781] To generate the desired siRNA duplex, two complementary strands were annealed by combining equimolar aqueous solutions of the two strands. The mixture was placed in a 70 °C water bath for 5 minutes and then cooled to ambient temperature over 2 hours. The duplex was lyophilized for 2 days and stored at -20 °C.

[1782] On a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system on a Superdex TM75Increase the 5 / 150GL column 5 x 153 - 158 mm (Cytiva) and analyze the duplex by analytical SEC HPLC. The mobile phase consists of 1x PBS containing 10% acetonitrile. At room temperature, run an isocratic gradient at a flow rate of 1.5 mL / min for 10 min. Record the UV traces at 260 and 280 nm. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[1783] Example 3: Synthesis of Tether 2

[1784] General experimental conditions:

[1785] Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using Macherey-Nagel's 254 nm fluorescent indicator. The compounds were visualized under ultraviolet light (254 nm), or sprayed with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to the method of Stahl (from Sigma-Aldrich) and then heated. Flash chromatography was carried out using a Biotage Isolera One flash chromatography instrument equipped with a bivariate UV wavelength detector (200 - 400 nm) and using Biotage 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1786] ...

Claims

1. A nucleic acid for inhibiting the expression of a target gene, comprising a duplex region, said duplex region comprising: a first strand that is at least partially complementary to a portion of the RNA transcribed from the target gene, and a second strand that is at least partially complementary to the first strand; wherein the nucleosides of the second strand and the first strand have the following 2'-sugar modification patterns (5'-3'): Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me or Second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me.

2. The nucleic acid according to claim 1, wherein the nucleosides of the second strand and the first strand have the following 2'-sugar and internucleoside bond modification patterns (5'-3'): Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me or Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where (s) is a phosphorothioate internucleoside bond.

3. The nucleic acid according to claim 1, wherein the nucleosides of the second strand and the first strand have the following 2'-sugar and abasic modification patterns (5'-3'): Second strand (5'-3'): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me, or Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me, wherein ia represents an inverted abasic nucleoside.

4. The nucleic acid according to claim 1, wherein the nucleosides of the second strand and the first strand comprise the following 2'-sugar, abasic, and linkage modification patterns (5’-3’): Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, or Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, wherein: (s) is a phosphorothioate internucleoside linkage, and ia represents an inverted abasic nucleoside.

5. The nucleic acid according to any one of the preceding claims, wherein the first strand comprises at least 17 consecutive nucleosides that differ from any one of the first strand sequences listed in Table 2 by 0 or 1 nucleoside.

6. The nucleic acid according to any one of the preceding claims, wherein the first strand comprises at least 17 consecutive nucleosides that differ from any one of the first strand sequences listed in Table 3 by 0 or 1 nucleoside.

7. The nucleic acid according to claim 5 or 6, wherein the first strand comprises nucleosides 2-18 of any one of the sequences defined in claim 5 or 6.

8. The nucleic acid according to any one of the preceding claims, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of the second strand sequences listed in Table 2 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand over the 17 consecutive nucleosides.

9. The nucleic acid according to any one of the preceding claims, wherein the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of the second strand sequences listed in Table 4 by 0 or 1 nucleoside, and wherein the second strand has a region that is at least 85% complementary to the first strand over the 17 consecutive nucleosides.

10. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is a siRNA oligonucleotide.

11. The nucleic acid according to any one of the preceding claims, wherein the nucleic acid is directly or indirectly conjugated to one or more ligand moieties, optionally, wherein the ligand moiety is present in the terminal region of the second strand, typically present in its 3' terminal region.

12. The nucleic acid according to claim 11, wherein the ligand moiety comprises: (iv) one or more N-acetylgalactosamine (GalNAc) ligands, and / or (v) one or more N-acetylgalactosamine (GalNAc) ligand derivatives, and / or (vi) one or more N-acetylgalactosamine (GalNAc) ligands and / or their derivatives, which are conjugated to the nucleic acid via a linker.

13. The nucleic acid according to claim 12, wherein the one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly conjugated to the 5' or 3' terminal region of the second strand of the nucleic acid, typically conjugated to its 3' terminal region.

14. The nucleic acid according to any one of claims 11-13, having the structure: wherein: R1 is independently selected from the group consisting of hydrogen, methyl and ethyl each time it appears; R2 is selected from the group consisting of: hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro; X1 and X2 are independently selected from the group consisting of methylene, oxygen and sulfur each time they appear; m is an integer from 1 to 6; n is an integer from 1 to 10; q, r, s, t, v are independently integers from 0 to 4, provided that: (i) q and r cannot be 0 simultaneously; and (ii) s, t and v cannot be 0 simultaneously; Z is an oligonucleotide.

15. The nucleic acid according to any one of claims 11-13, having the structure: wherein: r and s are independently integers selected from 1 to 16; and Z is an oligonucleotide.

16. A pharmaceutical composition comprising the nucleic acid according to any one of the preceding claims, and a pharmaceutically acceptable excipient or carrier.

17. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for treatment.

18. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for preventing or treating a disease associated with a hemostatic disorder, such as a disease associated with a hemostatic disorder (such as hemophilia).

19. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for preventing or treating cardiovascular diseases.

20. Use of the nucleic acid or pharmaceutical composition according to any one of the preceding claims for preventing or treating diabetes.

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