Nucleic acid compounds

Abstract

The present invention provides novel nucleic acid compounds suitable for therapeutic use. Additionally, the present invention provides methods for making these compounds and methods for using the compounds to treat various diseases and conditions.

Description

[Technical Field]

[0001] The present invention provides novel nucleic acid compounds suitable for therapeutic use. Additionally, the present invention provides methods for making these compounds and methods for using the compounds to treat various diseases and conditions. [Background technology]

[0002] Nucleic acid compounds have important therapeutic applications in medicine. Nucleic acids can be used to silence genes responsible for certain diseases. Gene silencing prevents the formation of proteins by inhibiting translation. Importantly, gene silencing agents 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 the formation of proteins through gene silencing.

[0003] Several modified siRNA compounds have been specifically developed over the past two decades for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutic agents for treating a variety of diseases, including diseases of the central nervous system, inflammatory diseases, metabolic disorders, oncology, infectious diseases, and ocular diseases. Summary of the Invention

[0004] The present invention relates to nucleic acid compounds for use in the treatment and / or prevention of disease.

[0005] Description of the Invention 1. A nucleic acid for inhibiting expression of a target gene, comprising: a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of said first strand comprise a 2' sugar modification pattern wherein said modifications are selected from at least 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of 4 or 6 2'F modifications.

[0006] The nucleic acid described herein, wherein the nucleosides of said first strand comprise a 2' sugar modification pattern, said modifications being selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5, or 7 2'F modifications.

[0007] The nucleic acid described herein, wherein the nucleosides of said first strand comprise a 2' sugar modification pattern, said modifications being selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of three 2'F modifications.

[0008] The nucleic acid described herein, wherein the nucleosides of said first strand comprise a 2' sugar modification pattern, said modifications being selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of five 2'F modifications.

[0009] The nucleic acid described herein, wherein the nucleosides of said first strand comprise a 2' sugar modification pattern, said modifications being selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of 7 2'F modifications.

[0010] The nucleosides of the first strand are (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 wherein X1 is a thermolabilizing modification. A nucleic acid as described herein, comprising a 2' sugar modification pattern of:

[0011] The nucleosides of the first strand are (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 wherein X2, X3, and X4 are selected from 2'Me and 2'F sugar modifications, provided that at least one of X2, X3, and X4 is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications; In particular, X2 is a 2'F sugar modification and X3 and X4 are 2'Me sugar modifications; or X3 is a 2'F sugar modification and X2 and X4 are 2'Me sugar modifications; or X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications. A nucleic acid as described herein, comprising a 2' sugar modification pattern of:

[0012] The nucleosides of the first strand are (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 wherein X1 is a thermolabilizing modification. A nucleic acid as described herein, comprising a 2' sugar modification pattern of:

[0013] The nucleosides of the first strand are (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 wherein X2, X3, and X4 are selected from 2'Me and 2'F sugar modifications, provided that at least one of X2, X3, and X4 is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications; In particular, X2 is a 2'F sugar modification and X3 and X4 are 2'Me sugar modifications; or X3 is a 2'F sugar modification and X2 and X4 are 2'Me sugar modifications; or X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications. A nucleic acid as described herein, comprising a 2' sugar modification pattern of:

[0014] A nucleic acid described herein, wherein two phosphorothioate internucleoside linkages are present between each of three consecutive positions in the 5'-terminal near region of the second strand, a first phosphorothioate internucleoside linkage is present between a first base nucleoside and an adjacent second base nucleoside in the 5'-terminal near region of the second strand, when read from the 5'-terminal, and a second phosphorothioate internucleoside linkage is present between the second base nucleoside and an adjacent third base nucleoside in the 5'-terminal near region of the second strand.

[0015] A nucleic acid as described herein, wherein two phosphorothioate internucleoside linkages are present between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, respectively, whereby the terminal nucleoside in each of the 5'-terminal region and the 3'-terminal region of the first strand is attached to each of the 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage, and each of the 5'- and 3'-adjacent penultimate nucleosides is attached to each of the 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage.

[0016] 1. A nucleic acid for inhibiting expression of a target gene, comprising: a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, where X1 is a thermolabilizing modification. (Me-F)3-(Me)7-F-Me-F-(Me)7 Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5 Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3 Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7, where X1 is a thermolabilizing modification. (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6 Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5 Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3 A nucleic acid comprising a 2' sugar modification pattern selected from one of:

[0017] 1. A nucleic acid for inhibiting expression of a target gene, comprising: a first strand that is at least partially complementary to a portion of RNA transcribed from the target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)(Me)3-X1-(Me)7-F-Me-F-(Me)5(s)Me(s)Me, where X1 is a thermolabilizing modification. Me(s)F(s)Me-F-Me-F-(Me)7-F-Me-F-(Me)5(s)Me(s)Me Me(s)F(s)(Me)3-F-(Me)7-(F-Me)2-F-(Me)3(s)Me(s)Me Me(s)F(s)(Me)3-F-(Me)7-F-Me-F-(Me)3-F-Me(s)Me(s)Me Me(s)F(s)(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)5(s)Me(s)Me, where X1 is a thermolabilizing modification. Me(s)F(s)Me-F-Me-F-Me-(F)2-(Me)4-(F-Me)2-(Me)4(s)Me(s)Me Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)3(s)Me(s)Me Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-Me(s)Me(s)Me A nucleic acid comprising a 2' sugar modification pattern selected from one of:

[0018] A nucleic acid as described herein, wherein two consecutive abasic nucleosides in the 5'-terminal region of the second strand include an abasic nucleoside that 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, and (a) the penultimate abasic nucleoside is connected to the adjacent first abasic nucleoside in the adjacent 5'-terminal proximal region through an inverted internucleoside linkage, (b) the inverted linkage is a 5-5' inverted linkage, and (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when reading toward the end that includes the terminal abasic nucleoside and the penultimate abasic nucleoside.

[0019] A nucleic acid according to the invention can further comprise a first strand comprising at least 17 consecutive nucleosides that differ by 0 or 1 nucleoside from any one of the first strand sequences listed in Table 2.

[0020] A nucleic acid according to the invention can further comprise a first strand comprising at least 17 consecutive nucleosides that differ by 0 or 1 nucleoside from any one of the first strand sequences listed in Table 3.

[0021] Typically, the first strand comprises nucleosides 2 to 18 of any one of the sequences defined in Tables 2 or 3.

[0022] A nucleic acid according to the invention may further comprise a second strand comprising a nucleoside sequence of at least 17 consecutive nucleosides that differs by 0 or 1 nucleoside from any one of the second strand sequences listed in Table 2, wherein the duplex region comprises at least 14, 15, 16, or 17 complementary base pairs.

[0023] A nucleic acid according to the invention may further comprise a second strand comprising a nucleoside sequence of at least 17 consecutive nucleosides that differs by 0 or 1 nucleoside from any one of the second strand sequences listed in Table 4, wherein the duplex region comprises at least 14, 15, 16, or 17 complementary base pairs.

[0024] A nucleic acid according to the invention, wherein the first strand comprises any one of the first strand sequences listed in Table 2.

[0025] A nucleic acid according to the invention, wherein the first strand comprises any one of the first strand sequences listed in Table 3.

[0026] A nucleic acid according to the invention, wherein the second strand comprises any one of the second strand sequences listed in Table 2.

[0027] A nucleic acid according to the invention, wherein the second strand comprises any one of the second strand sequences listed in Table 4.

[0028] A nucleic acid according to the invention, wherein the first strand and the second strand form any one of the duplexes listed in Table 5.

[0029] The nucleic acid according to the invention, wherein the nucleic acid is an siRNA oligonucleoside.

[0030] A nucleic acid according to the present invention, wherein the nucleic acid is directly or indirectly conjugated to one or more ligand moieties, optionally located at the terminal region of the second strand, typically at its 3'-terminal region, and typically comprising one or more N-acetylgalactosamine (GalNAc) ligands and / or one or more N-acetylgalactosamine (GalNAc) ligand derivatives, and / or one or more N-acetylgalactosamine (GalNAc) ligands and / or derivatives thereof conjugated to the nucleic acid via a linker. Typically, the one or more GalNAc ligands and / or GalNAc ligand derivatives are conjugated directly or indirectly to the 5'- or 3'-terminal region of the second strand of the nucleic acid, typically at its 3'-terminal region.

[0031] The following structure:

[0032] [ka]

[0033] A nucleic acid according to the present invention comprising a ligand moiety comprising:

[0034] The following structure:

[0035] [ka]

[0036] [In the formula, R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl, and ethyl; R2 is hydrogen, hydroxy, -OC 1~3 Alkyl, -C(=O)OC 1~3 selected from the group consisting of alkyl, halo, and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen, and sulfur; m is an integer from 1 to 6, n is an integer from 1 to 10, q, r, s, t, and v are independently integers from 0 to 4, provided that q and r cannot both be 0 at the same time, and s, t, and v cannot all be 0 at the same time; Z is an oligonucleoside. A nucleic acid according to the present invention comprising a ligand moiety comprising:

[0037] structure

[0038] [ka]

[0039] wherein [oligonucleotide] represents consecutive nucleosides of the second strand. The nucleic acid according to the present invention, comprising:

[0040] Or the following structure:

[0041] [ka]

[0042] [In the formula, r and s are independently an integer selected from 1 to 16; Z is an oligonucleoside. A nucleic acid according to the present invention comprising a ligand moiety comprising:

[0043] structure

[0044] [ka]

[0045] wherein [oligonucleotide] represents consecutive nucleosides of the second strand. The nucleic acid according to the present invention, comprising:

[0046] The present invention further provides pharmaceutical compositions comprising the nucleic acids described herein in combination with a pharmaceutically acceptable excipient or carrier.

[0047] The present invention further provides a nucleic acid or pharmaceutical composition described herein for use in therapy.

[0048] The invention further provides a nucleic acid or pharmaceutical composition as described herein for use in the prevention or treatment of a disease associated with impaired hemostasis, for example a disease associated with impaired hemostasis such as hemophilia.

[0049] The present invention further provides a nucleic acid or pharmaceutical composition described herein for use in the prevention or treatment of diabetes.

[0050] The present invention further provides a nucleic acid or pharmaceutical composition described herein for use in the prevention or treatment of cardiovascular disease. [Brief explanation of the drawings]

[0051] [Figure 1]

[0023] Figure 1 shows a linker to be conjugated to an oligonucleotide, but it should be understood that the present invention also encompasses conjugations of the same linker with an oligonucleoside disclosed herein. While Figure 1 shows as a product a molecule based on the linker and ligand moieties specifically shown in Figure 1 attached to an oligonucleoside moiety also shown herein, it should also be understood that the product may alternatively further comprise or consist essentially of a molecule in which the linker and ligand moieties are attached to an oligonucleoside moiety essentially as shown in Figure 1, but in which the F substituent shown in Figure 1 on the cyclo-octyl ring has been replaced by a substituent resulting from a hydrolyzable substitution, such as an OH substituent. In this manner, (a) Tether 1a constructs can consist essentially of molecules having linker and ligand moieties as specifically shown in FIG. 1 with an F substituent on the cyclo-octyl ring; or (b) Tether 1a constructs can consist essentially of molecules having linker and ligand moieties as specifically shown in FIG. 1, but in which the F substituent shown in FIG. 1 on the cyclo-octyl ring is replaced by a substituent resulting from a hydrolyzable substitution, such as an OH substituent; or (c) Tether 1a constructs can comprise a mixture of molecules as defined in (a) and / or (b). [Figure 2]Linker and Ligand Moieties of Constructs Suitable for Use According to the Invention Comprising Tether 1b. While Figure 2 shows a linker to be conjugated to an oligonucleotide, it should be understood that the invention also encompasses conjugation of the same linker with the oligonucleosides disclosed herein. The comments made in connection with Figure 1 and the possible replacement of the F substituent shown in Figure 1 on the cyclo-octyl ring with a substituent resulting from hydrolyzable substitution, such as an OH substituent, apply equally to Tether 1b constructs. Thus, (a) Tether 1b constructs can consist essentially of molecules having linker and ligand moieties specifically shown in Figure 2 with an F substituent on the cyclo-octyl ring, or (b) Tether 1b constructs can consist essentially of molecules having linker and ligand moieties as shown in Figure 2, but with the F substituent shown in Figure 2 on the cyclo-octyl ring replaced by a substituent resulting from hydrolyzable substitution, such as an OH substituent, or (c) Tether 1b constructs can comprise a mixture of molecules defined in (a) and / or (b). [Figure 3] Linker and Ligand Moieties of Constructs Suitable for Use According to the Invention Comprising Tether 2a. While Figure 3 shows the linker to be conjugated to an oligonucleotide, it should be understood that the invention also encompasses conjugations of the same linker with the oligonucleosides disclosed herein. [Figure 4] Linker and Ligand Moieties of Constructs Suitable for Use According to the Invention Comprising Tether 2b. While Figure 4 shows the linker to be conjugated to an oligonucleotide, it should be understood that the invention also encompasses conjugations of the same linker with the oligonucleosides disclosed herein. [Figure 5-1] The formulas described in sentences 1 to 101 of this specification. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-2. [Figure 5-4] This is a continuation of Figure 5-3. [Figure 5-5]This is a continuation of Figure 5-4. [Figure 5-6] This is a continuation of Figure 5-5. [Figure 5-7] This is a continuation of Figure 5-6. [Figure 5-8] This is a continuation of Figure 5-7. [Figure 6-1] The formulas set forth in paragraphs 1 to 56 of the present specification. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 6-3] This is a continuation of Figure 6-2. [Figure 6-4] This is a continuation of Figure 6-3. [Figure 6-5] This is a continuation of Figure 6-4. [Figure 7] Exemplary modification patterns within the scope of the present invention. The iaia shown in the 5'-terminal region of the sense strand (i) represents two abasic nucleosides provided as the penultimate nucleoside and terminal nucleoside in the 5'-terminal region of the sense strand, (ii) a 5'-5' reverse linkage is provided between the penultimate nucleoside of the sense strand (i.e., at position 1 of the sense strand, which does not include the iaia motif in the 5'-terminal region of the sense strand at the nucleoside position numbered on the sense strand) and the adjacent penultimate abasic residue, and (iii) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when reading toward the 5'-terminal region including the terminal abasic nucleoside and the penultimate abasic nucleoside. Nucleosides with 2'-O-Me modifications are designated "Me." Nucleosides with 2'-F modifications are designated "F." Nucleosides with either a 2'-O-Me or a 2'-F modification are designated "Me / F." Exemplary modification patterns can be applied to nucleic acid sequences according to the invention described herein. A GalNAc linker may be attached to the 3'-terminal region of the sense strand used (not shown in Figure 7). [Figure 8]Inhibition of ZPI expression by ETXM1200 (ETXS2400 and ETXS2397), ETXM1203 (ETXS2406 and ETXS2397), ETXM1204 (ETXS2408 and ETXS2397), ETXM1205 (ETXS2410 and ETXS2397), ETXM1206 (ETXS2412 and ETXS2397), and ETXM1207 (ETXS2414 and ETXS2397). [Figure 9] Inhibition of B4GALT1 expression by ETXM1217 (ETXS2434 and ETXS2401), ETXM1766 (ETXS3532 and ETXS2401), ETXM1767 (ETXS3534 and ETXS2401), ETXM1768 (ETXS3536 and ETXS2401), ETXM1769 (ETXS3538 and ETXS2401), and ETXM1770 (ETXS3540 and ETXS2401). [Figure 10] Inhibition of B4GALT1 expression by ETXM1218 (ETXS2436 and ETXS2407), ETXM1774 (ETXS3548 and ETXS2407), ETXM1775 (ETXS3550 and ETXS2407), ETXM1776 (ETXS3552 and ETXS2407), ETXM1777 (ETXS3554 and ETXS2407), and ETXM1778 (ETXS3556 and ETXS2407). DETAILED DESCRIPTION OF THE INVENTION

[0052] definition "First strand", also referred to herein as antisense strand or guide strand, and interchangeably used herein, refers to the strand of nucleic acid, for example, siRNA, for example, dsiRNA, that comprises a region that is substantially complementary to target sequence, for example, mRNA.As used herein, the term "complementary region" refers to the region on the antisense strand that is substantially complementary to sequence, for example, target sequence.If the complementary region is not completely complementary to the target sequence, mismatch may typically be in the internal or terminal region of the molecule.In some embodiments, the double-stranded nucleic acid of the present invention, for example, siRNA agent, comprises nucleoside mismatch in the antisense strand.

[0053] "Second strand" (also referred to herein as the sense strand or passenger strand, and may be used interchangeably herein) refers to the strand of a nucleic acid, e.g., an siRNA, that includes a region that is substantially complementary to a region of the antisense strand, as defined herein.

[0054] In the context of molecules comprising a nucleic acid with a ligand moiety, and optionally with a linker moiety, the nucleic acids of the invention can be referred to as oligonucleosides or oligonucleoside moieties.

[0055] Oligonucleotides are short nucleic acid polymers. Although oligonucleotides contain phosphodiester bonds between their nucleoside components (base + sugar), the present invention is not limited to oligonucleotides always linked by such phosphodiester bonds between adjacent nucleosides; other oligomers of nucleosides linked by bonds other than phosphodiester bonds are contemplated. For example, the internucleoside bond may be a phosphorothioate bond. Thus, the term "oligonucleoside" as used herein covers both oligonucleotides and other oligomers of nucleosides. Oligonucleosides that are nucleic acids with at least one portion being an oligonucleotide are preferred according to the present invention. Oligonucleosides with one or more, or even a majority of, phosphodiester backbone bonds between nucleosides are also preferred according to the present invention. Oligonucleosides having one or more, or a majority of, phosphodiester backbone linkages between nucleosides and also having one or more phosphorothioate backbone linkages between nucleosides (typically in the terminal regions of the first and / or second strands) are also preferred in accordance with the present invention.

[0056] It is preferred herein that the nucleic acid according to the present invention is a double-stranded oligonucleoside comprising one or more phosphorothioate backbone bonds between nucleosides.Therefore, in all cases where the present application refers to an oligonucleotide, particularly in the chemical structure disclosed herein, the oligonucleotide may also be an oligonucleoside as defined herein.

[0057] In some embodiments, the double-stranded nucleic acids, e.g., siRNA agents, of the invention contain a nucleoside mismatch in the sense strand, e.g., within 5, 4, 3, 2, or 1 nucleoside from the 3' end of the nucleic acid, e.g., siRNA.

[0058] In another embodiment, the nucleoside mismatch is at the 3' terminal nucleoside of the nucleic acid, eg, the siRNA.

[0059] "Target sequence" (also called target RNA or target mRNA) refers to a contiguous portion of the nucleoside sequence of an mRNA molecule formed during transcription of a gene, including mRNA that is the product of RNA processing of the primary transcription product.

[0060] The target sequence may be about 10 to 35 nucleosides in length, for example, about 15 to 30 nucleosides in length. For example, the target sequence may be about 15 to 30 nucleosides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 30 ... The length may be up to 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 nucleosides. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the invention.

[0061] The terms "ribonucleoside" or "nucleoside" can also refer to modified nucleosides, which are described in more detail below.

[0062] The nucleic acid may be DNA or RNA and may contain modified nucleosides. RNA is the preferred nucleic acid.

[0063] The terms "iRNA," "siRNA," "RNAi agent," and "iRNA agent," "RNA interfering agent," when used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. siRNA directs the sequence-specific degradation of mRNA through RNA interference (RNAi).

[0064] Double-stranded RNA, referred to herein as a "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," refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands said to have "sense" and "antisense" orientations relative to a target RNA.

[0065] The majority of nucleosides in each strand of a nucleic acid, e.g., a dsiRNA molecule, are preferably ribonucleosides, although in such cases, each or both strands may also contain one or more non-ribonucleosides, e.g., deoxyribonucleosides or modified nucleosides. Furthermore, as used herein, "siRNA" may include ribonucleosides having chemical modifications.

[0066] The term "modified nucleoside" refers to a nucleoside having, independently, a modified sugar moiety, a modified internucleoside linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleoside encompasses the substitution, addition, or removal of, for example, a functional group or atom, to an internucleoside linkage, a sugar moiety, or a nucleobase. Any such modifications are encompassed by "iRNA" or "RNAi agent" or "siRNA" or "siRNA agent" for purposes of this specification and claims when used in siRNA-type molecules.

[0067] The two strands forming the duplex structure may be different parts of one larger molecule, or they may be separate molecules, such as RNA molecules.

[0068] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the duplex structure of a nucleic acid according to the present invention. A nucleic acid according to the present invention may comprise an overhang of at least one nucleoside; alternatively, the overhang may comprise at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more. The nucleoside overhang may comprise or consist of nucleoside / nucleoside analogs, including deoxynucleosides. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleoside(s) of the overhang may be present on the 5'-end, the 3'-end, or both ends of either the antisense or the sense strand.

[0069] In certain embodiments, the antisense strand has an overhang of 1 to 10 nucleosides at the 3' or 5' end, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides.

[0070] "Blunt" or "blunt end" means that there are no unpaired nucleosides at the ends of a double-stranded nucleic acid, i.e., there are no nucleoside overhangs. Nucleic acids of the present invention include those that have no nucleoside overhangs at one end or neither end.

[0071] Unless otherwise indicated, the term "complementary," when used to describe a first nucleoside sequence in relation to a second nucleoside sequence, refers to the ability of an oligonucleoside comprising the first nucleoside sequence to hybridize to an oligonucleoside comprising the second nucleoside sequence under certain conditions to form a duplex structure, as will be understood by those of skill in the art. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press).

[0072] As used herein, a complementary sequence in a nucleic acid, such as a dsiRNA, includes base pairing of an oligonucleoside containing a first nucleoside sequence with an oligonucleoside containing a second nucleoside sequence across the entire length of one or both nucleoside sequences. Such sequences can be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "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, such as 2, 4, or 5, but preferably no more than 5, mismatched base pairs, while maintaining the ability to hybridize under conditions most relevant to the final application, such as inhibiting gene expression via the RISC pathway. Overhangs are not considered mismatches when determining complementarity. For example, a nucleic acid, e.g., a dsiRNA, comprising one oligonucleoside 17 nucleosides in length and another oligonucleoside 19 nucleosides in length, where the longer oligonucleoside comprises a sequence of 17 nucleosides that is perfectly complementary to the shorter oligonucleoside, can still be said to be "fully complementary."

[0073] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleosides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0074] The terms "complementary," "fully complementary," and "substantially / partially complementary" herein can be used in reference to base matching between a nucleic acid, e.g., the sense strand and antisense strand of a dsiRNA, or between a double-stranded nucleic acid, e.g., the antisense strand of an siRNA agent, and a target sequence.

[0075] In the present invention, the second strand of a nucleic acid according to the present invention is at least partially complementary to the first strand of said nucleic acid. In certain embodiments, the first and second strands of a nucleic acid according to the present invention are partially complementary if they form a duplex region that is at least 17 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0076] In certain embodiments, the first and second strands of a nucleic acid according to the invention are partially complementary if they form a duplex region that is 19 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, the first and second strands of a nucleic acid according to the invention are partially complementary if they form a duplex region that is 21 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0077] Alternatively, the first and second strands of a nucleic acid according to the invention are partially complementary if they form a duplex region having a length of at least 17 base pairs, at least 14, 15, 16 or 17 of which are complementary base pairs, in particular Watson-Crick base pairs.

[0078] In certain embodiments, the first and second strands of a nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 19 base pairs, and at least 14, 15, 16, 17, 18, or all 19 base pairs are complementary base pairs, particularly Watson-Crick base pairs. In certain embodiments, the first and second strands of a nucleic acid according to the invention are partially complementary if they form a duplex region having a length of 21 base pairs, and at least 16, 17, 18, 19, 20, or all 21 base pairs are complementary base pairs, particularly Watson-Crick base pairs.

[0079] As used herein, a nucleic acid that is "substantially complementary" or "partially complementary" to at least a portion of a messenger RNA (mRNA) refers to a nucleic acid that is substantially or partially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding a gene). In certain embodiments, the contiguous portion of the mRNA is any one of the sequences listed in Table 1, i.e., SEQ ID NOS: 4-17. For example, a nucleic acid is complementary to at least a portion of an mRNA of a gene of interest if the sequence is substantially or partially complementary to an uninterrupted portion of the mRNA encoding that gene.

[0080] Thus, in some preferred embodiments, the antisense oligonucleosides disclosed herein are perfectly complementary to the target gene sequence.

[0081] In other embodiments, the antisense oligonucleosides disclosed herein are substantially or partially complementary to a target RNA sequence, and comprise a contiguous nucleoside sequence that is at least about 80% complementary, e.g., at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, to the corresponding region of the target RNA sequence over its entire length.

[0082] In some embodiments, the nucleic acid of the present invention, for example, siRNA, comprises a sense strand that is substantially or partially complementary to antisense oligonucleosides, and this antisense oligonucleoside is in turn complementary to target gene sequence and comprises a continuous nucleoside sequence.The nucleoside sequence of the sense strand is typically at least about 80% complementary to the equivalent region of the nucleoside sequence of antisense strand over its entire length, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0083] In some embodiments, the nucleic acids, e.g., siRNAs, of the invention comprise an antisense strand that is substantially or partially complementary to a target sequence and comprises a contiguous nucleoside sequence that is at least 80% complementary to the target sequence over its entire length, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.

[0084] As used herein, a "subject" is an animal, e.g., a mammal, such as a primate (e.g., a human, a non-human primate, e.g., a monkey or chimpanzee), or a non-primate or bird, that expresses a target gene, either endogenously or heterologously, where the target gene sequence has sufficient complementarity with a nucleic acid, e.g., an siRNA agent, to promote target knockdown. In certain preferred embodiments, the subject is a human.

[0085] The terms "treating" or "treatment" refer to a beneficial or desired result, including, but not limited to, alleviating or ameliorating one or more symptoms associated with gene expression. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Treatment can also include preventing the development of comorbidities, for example, reducing liver damage in a subject with a liver infection.

[0086] A "therapeutically effective amount," as used herein, is intended to include the amount of a nucleic acid, e.g., an siRNA, that, when administered to a patient for treating a subject having a disease, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease or its associated comorbidities).

[0087] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0088] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated.

[0089] When a value or range of values for a parameter is listed, it is intended that values and ranges intermediate to the listed values are also intended to be part of the invention.

[0090] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0091] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0092] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. For example, "the sense strand or the antisense strand" is understood as "the sense strand or the antisense strand, or the sense strand and the antisense strand."

[0093] The term "about" is used herein to mean within typical tolerances in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is present before a series of numbers or ranges, it is understood that "about" can modify each of the numbers in the series or range.

[0094] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that can be logically included, as is clear from the context. For example, the number of nucleosides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides of a 21-nucleoside nucleic acid molecule" means that 18, 19, 20, or 21 nucleosides have the specified property. When at least is present before a series or range of numbers, it is understood that "at least" can modify each of the numbers in the series or range.

[0095] As used herein, "not more than" or "less than" shall be understood to refer to the value adjacent to the phrase and the logical lower value or integer up to zero, as is logical from the context. For example, a duplex having "not more than two nucleosides" overhangs will have 2, 1, or 0 nucleoside overhangs. When "not more than" precedes a series of numbers or ranges, it is understood that "not more than" can modify each of the numbers in the series or range.

[0096] The terminal region of the chain is the last five nucleosides from the 5' or 3' end.

[0097] The various embodiments of the invention may be combined as determined appropriate by those skilled in the art.

[0098] Abasic nucleosides In certain embodiments, one, e.g., two, e.g., three, e.g., four, or more abasic nucleosides are present in a nucleic acid according to the present invention. Abasic nucleosides are modified nucleosides because they lack the base normally found at position 1 of the sugar moiety. Typically, there is a hydrogen at position 1 of the sugar moiety of abasic nucleosides present in a nucleic acid according to the present invention.

[0099] The abasic nucleoside is in a terminal region of the second strand, preferably located within the terminal 5 nucleosides of the end of the strand. The terminal region may be the terminal 5 nucleosides that include the abasic nucleoside.

[0100] The second strand may include the following as preferred features (all of which are specifically contemplated in combination, unless mutually exclusive): two or more abasic nucleosides in the terminal region of the second strand, and / or two or more abasic nucleosides in either the 5' or 3' terminal region of the second strand, and / or two or more abasic nucleosides in either the 5' or 3' terminal region of the second strand, wherein the abasic nucleosides are present in an overhang as described herein; and / or two or more consecutive abasic nucleosides in the terminal region of the second strand, preferably one of the abasic nucleosides being the terminal nucleoside; and / or two or more consecutive abasic nucleosides in either the 5' or 3' terminal region of the second strand, preferably one of the abasic nucleosides being the terminal nucleoside in either the 5' or 3' terminal region of the second strand; and / or an inverted internucleoside linkage connects at least one abasic nucleoside to an adjacent basic nucleoside in the terminal region of the second strand; and / or an inverted internucleoside linkage connects at least one abasic nucleoside to an adjacent basic nucleoside in either the 5' or 3' terminal region of the second strand; and / or an abasic nucleoside as the penultimate nucleoside connected via a retrograde linkage to a nucleoside that is not the terminal nucleoside (referred to herein as the penultimate nucleoside); and / or an abasic nucleoside as the two terminal nucleosides connected via a 5'-3' linkage when the chain is read in the direction toward the end containing the terminal nucleoside; an abasic nucleoside as the two terminal nucleosides connected via a 3'-5' linkage when the chain is read in the direction toward the end containing the terminal nucleoside; abasic nucleosides as the terminal two positions, wherein the penultimate nucleoside is connected to the penultimate nucleoside via an inverted linkage, the inverted linkage being a 5-5' inverted linkage or a 3'-3' inverted linkage; Abasic nucleosides as the terminal two positions, where the penultimate nucleoside is connected to the penultimate nucleoside via a retrograde linkage, and is either: (1) the inverted linkage is a 5-5' inverted linkage, and the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'5' when read toward the end containing the terminal abasic nucleoside and the penultimate abasic nucleoside; or (2) The inverted linkage is a 3-3' inverted linkage, in which the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 5'3' when read toward the end containing the terminal abasic nucleoside and the penultimate abasic nucleoside.

[0101] Preferably, there is an abasic nucleoside at the terminus of the second strand.

[0102] Preferably, there are two or at least two abasic nucleosides in the terminal regions of the second strand, preferably in the terminal and penultimate positions.

[0103] Preferably, two or more abasic nucleosides are contiguous, and for example, all of the abasic nucleosides may be contiguous, for example, the terminal one, two, three, or four nucleosides may be abasic nucleosides.

[0104] An abasic nucleoside may also be linked to an adjacent nucleoside through a 5'-3' phosphodiester linkage or a reverse linkage, except when there is only one abasic nucleoside at the terminal end, in which case it has a reverse linkage to the adjacent nucleoside.

[0105] An inverted linkage (which may also be referred to as an inverted linkage and is also found in the art) comprises either a 5'-5', 3'3', 3'-2' or 2'-3' phosphodiester linkage between adjacent sugar moieties of the nucleoside.

[0106] The non-terminal abasic nucleoside has two phosphodiester bonds with each adjacent nucleoside, which may be inverted or 5'-3 phosphodiester bonds, or one of each.

[0107] A preferred embodiment comprises two abasic nucleosides at the terminal and penultimate positions of the second strand, with the reverse internucleoside linkage being located between the penultimate (abasic) nucleoside and the penultimate nucleoside.

[0108] Preferably, there are two abasic nucleosides at the terminal and penultimate positions of the second strand, with the penultimate nucleoside linked to the penultimate nucleoside through a reverse internucleoside linkage and linked to the terminal nucleoside through a 5'-3' or 3'-5' phosphodiester linkage (reading towards the ends of the molecule).

[0109] Preferably, the nucleic acid according to the present invention comprises one or more abasic nucleosides, optionally one or more abasic nucleosides in the terminal region of the second strand, and / or at least one abasic nucleoside is linked to an adjacent abasic nucleoside via a reverse internucleoside linkage.

[0110] Typically, the second strand comprises two consecutive abasic nucleosides in the 5'-terminal region of the second strand, one abasic nucleoside 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, (a) the penultimate abasic nucleoside is connected to the adjacent first abasic nucleoside in the adjacent 5'-terminal proximal region through an inverted internucleoside linkage, (b) the inverted linkage is a 5-5' inverted linkage, and (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'5' when read toward the end comprising the terminal abasic nucleoside and the penultimate abasic nucleoside. More typically, (i) the first strand and the second strand each have a length of 23 nucleosides, (ii) two phosphorothioate internucleoside linkages are respectively between three consecutive positions in the 5'-terminal proximal region of the second strand, with a first phosphorothioate internucleoside linkage being between the adjacent first base nucleoside and the adjacent second base nucleoside of (a) in the 5'-terminal proximal region of the second strand, and a second phosphorothioate internucleoside linkage being between the adjacent second base nucleoside and the adjacent third base nucleoside in the 5'-terminal proximal region of the second strand, and (iii) two phosphorothioate internucleoside linkages are The linkages are between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, respectively, whereby the terminal nucleoside in each of the 5'-terminal region and the 3'-terminal region of the first strand is attached to each of the 5'- and 3'-adjacent penultimate nucleosides, respectively, by a phosphorothioate internucleoside linkage, and each of the first 5'- and 3'-adjacent penultimate nucleosides is attached to each of the 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage, and (iv) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties at the 3'-terminal region of the second strand.

[0111] Exemplary structures are as follows (the specific RNA nucleosides shown are not limiting and can be any RNA nucleoside):

[0112] A 3'-3' reverse bond (reading toward the end of the molecule, also showing the 5'-3 orientation of the final phosphodiester bond between two abasic molecules)

[0113] [ka]

[0114] B illustrates a 5'-5' reverse bond (reading toward the end of the molecule, also showing the 3'-5' orientation of the final phosphodiester bond between two abasic molecules)

[0115] [ka]

[0116] The abasic nucleoside(s) present in the nucleic acid are provided in the presence of inverted internucleoside linkage(s), i.e., 5'-5' or 3'-3' inverted internucleoside linkage. Inverted linkages arise as a result of a change in the orientation of adjacent nucleoside sugars, such that the sugars have a 3'-5' orientation as opposed to the conventional 5'-3' orientation (with reference to the numbering of the ring atoms on the nucleoside sugar). The abasic nucleoside(s) present in the nucleic acids of the present invention preferably comprise such inverted nucleoside sugars.

[0117] In the case of a terminal nucleoside having an inverted orientation, this results in a "flipped" terminal configuration for the overall nucleic acid. While certain structures depicted and referenced herein are represented using the conventional 5'-3' orientation (with reference to the numbering of the ring atoms on the nucleoside sugar), it is understood that a change in orientation and the presence of a terminal nucleoside having a proximal 3'-3' inverted linkage will result in a nucleic acid having an overall 5'-5' terminal structure (i.e., a conventional 3' terminal nucleoside becomes a 5' terminal nucleoside). Alternatively, it is understood that a change in orientation and the presence of a terminal nucleoside having a proximal 5'-5' inverted linkage will result in a nucleic acid having an overall 3'-3' terminal structure.

[0118] The proximal 3'-3' or 5'-5' inverted linkages described herein can include an inverted linkage directly adjacent to / attached to a terminal nucleoside having an inverted orientation, e.g., a single terminal nucleoside having an inverted orientation. Alternatively, the proximal 3'-3' or 5'-5' inverted linkages described herein can include inverted linkages of two or more adjacent nucleosides having an inverted orientation, e.g., two or more terminal region nucleosides having an inverted orientation, e.g., a terminal nucleoside and a penultimate nucleoside. In this manner, the inverted linkage can be attached to a penultimate nucleoside having an inverted orientation. Those skilled in the art will understand that the above inverted orientations may result in nucleic acid molecules having the overall 3'-3' or 5'-5' end structures described herein, but will also understand that the presence of one or more additional inverted linkages and / or nucleosides having an inverted orientation may result in the overall nucleic acid having a 3'-5' end structure corresponding to the conventionally arranged 5' / 3' ends.

[0119] In one aspect, the nucleic acid may have a 3'-3' inverted linkage and the terminal sugar moiety may comprise a 5' OH rather than a 5' phosphate group at the 5' position of the terminal sugar.

[0120] Thus, one of skill in the art will clearly understand that, when a reverse linkage(s) is / are present, 5'-5', 3'-3', and 3'-5' (reading towards the end) terminal variants of the more conventional 5'-3' structure (with reference to the numbering of the ring atoms on the terminal nucleoside sugar) shown herein are included within the scope of the present disclosure.

[0121] For example, in the context of one or more nucleosides having a reverse internucleoside linkage and / or a reversed orientation resulting in an inverted terminus, and when the relative position of the linkage (e.g., to a linker) or the position of an internal feature (e.g., a modified nucleoside) is 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 exist if the reverse linkage were not in place, and the conventional 5' or 3' end is determined by considering the orientation and / or nucleoside orientation of the majority of internal nucleoside linkages within the nucleic acid. From these internal linkages and / or nucleoside orientations, it is possible to identify which ends of the nucleic acid would constitute the conventional 5' and 3' ends (with reference to the numbering of the ring atoms on the terminal nucleoside sugar) of the molecule without the reverse linkage.

[0122] For example, in the structure shown below, there are abasic residues at the first two positions, located at the 5' end. If the terminal nucleoside has an inverted orientation, the 5' end shown in the schematic below, which is the conventional 5' end, may actually contain a 3' OH, taking into account the inverted nucleoside at the terminal position. Nevertheless, the majority of the molecules, when read in the standard 5'[PO4] to 3'[OH] direction of a nucleic acid molecule (with reference to the numbering of the ring atoms on the nucleoside sugar), contain conventional internucleoside linkages running from the 3' OH of one 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 the inverted terminal configuration.

[0123] 5'AA-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me 3'

[0124] The reverse bond is preferably located at the end of the nucleic acid, eg, RNA, distal to the ligand portion of the molecule, eg, the GalNAc-containing portion.

[0125] A GalNAc-siRNA construct having a 5'-GalNAc on the sense strand can have a reverse linkage on the opposite end of the sense strand.

[0126] A GalNAc-siRNA construct having a 3'-GalNAc on the sense strand can have a reverse linkage on the opposite end of the sense strand.

[0127] In certain embodiments, the present invention provides 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. A nucleic acid for inhibiting expression of a target gene, comprising a double-stranded region comprising: the second strand comprises two consecutive abasic nucleosides in the 5'-terminal region of the second strand, one of the abasic nucleoside 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; (a) the penultimate abasic nucleoside is connected to the adjacent first abasic nucleoside in the adjacent 5'-proximal region through a reverse internucleoside linkage; (b) the reverse ligation is a 5-5' reverse ligation; (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when read toward the end including the terminal abasic nucleoside and the penultimate abasic nucleoside; Concerning nucleic acids.

[0128] In certain embodiments, the present invention provides 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. A nucleic acid for inhibiting expression of a target gene, comprising a double-stranded region comprising: (i) preferably, the first strand and the second strand each have a length of 23 nucleosides (this length for the second strand includes two abasic nucleosides); (ii) the second strand comprises two consecutive abasic nucleosides in the 5'-terminal region of the second strand, one of the 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; (a) the penultimate abasic nucleoside is connected to the adjacent first abasic nucleoside in the adjacent 5'-proximal region through a reverse internucleoside linkage; (b) the reverse ligation is a 5-5' reverse ligation; (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when read toward the end including the terminal abasic nucleoside and the penultimate abasic nucleoside; (iii) two phosphorothioate internucleoside linkages are present between three consecutive positions in the 5'-terminal proximal region of the second strand, respectively, a first phosphorothioate internucleoside linkage is present between the first base nucleoside of (a) and the adjacent second base nucleoside in the 5'-terminal proximal region of the second strand, and a second phosphorothioate internucleoside linkage is present between the second base nucleoside and the adjacent third base nucleoside in the 5'-terminal proximal region of the second strand; (iv) two phosphorothioate internucleoside linkages are present between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, respectively, whereby the terminal nucleoside in each of the 5'-terminal region and the 3'-terminal region of the first strand is attached to each of the 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage, and each of the 5'- and 3'-adjacent penultimate nucleosides is attached to each of the 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage; (v) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties at the 3'-terminal region of the second strand; Concerning nucleic acids.

[0129] In certain embodiments, the present invention provides 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. A nucleic acid for inhibiting expression of a target gene, comprising a double-stranded region comprising: The second strand has the following 5' end motif:

[0130] [ka]

[0131] [In the formula, B represents a nucleoside base; T represents H, OH or a 2' ribose modification; Z represents the remaining nucleosides of the second strand. The present invention relates to a nucleic acid comprising two consecutive abasic nucleosides in the 5'-terminal region of the second strand, present as:

[0132] In certain embodiments, the present invention provides 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. A nucleic acid for inhibiting expression of a target gene, comprising a double-stranded region comprising: The second strand has the following 5' end motif:

[0133] [ka]

[0134] [In the formula, B represents a nucleoside base; T represents H, OH or a 2' ribose modification; V represents O or S (preferably O), R is H or C 1~4 represents alkyl (preferably H), Z represents the remaining nucleosides of said second strand; More preferably, the following 5'-end motif:

[0135] [ka]

[0136] [In the formula, B represents a nucleoside base; T represents H, OH or a 2' ribose modification; Z represents the remaining nucleosides of the second strand. The present invention relates to a nucleic acid comprising two consecutive abasic nucleosides in the 5'-terminal region of the second strand, present as:

[0137] In certain embodiments, the present invention provides 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. A nucleic acid for inhibiting expression of a target gene, comprising a double-stranded region comprising: The second strand has the following 5' end motif:

[0138] [ka]

[0139] [In the formula, B represents a nucleoside base; T represents H, OH or a 2' ribose modification; V represents O or S (preferably O), R is H or C 1~4 represents alkyl (preferably H), Z comprises 11 to 26 consecutive nucleosides, preferably 15 to 21 consecutive nucleosides, more preferably 19 consecutive nucleosides; More preferably, the following 5'-end motif:

[0140] [ka]

[0141] [In the formula, B represents a nucleoside base; T represents H, OH or a 2' ribose modification; Z comprises 11 to 26 consecutive nucleosides, preferably 15 to 21 consecutive nucleosides, more preferably 19 consecutive nucleosides. The present invention relates to a nucleic acid comprising two consecutive abasic nucleosides in the 5'-terminal region of the second strand, present as:

[0142] In some embodiments, the modification pattern of the second (sense) strand of a nucleic acid according to the present invention is i-i-a-Me-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, where i-a represents an inverted abasic nucleoside. It comprises or consists of:

[0143] In such embodiments, the second strand preferably has the following 5' end motif:

[0144] [ka]

[0145] [In the formula, B represents the nucleoside base of the first base nucleoside in the 5'-terminal region of the second strand; T represents a 2'Me ribose modification; Z represents the remaining consecutive base nucleosides of the second strand. Includes:

[0146] In some embodiments, the modification pattern of the second (sense) strand of a nucleic acid according to the present invention is i-i-a-Me(s)-Me(s)-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, where (s) is a phosphorothioate internucleoside linkage and i-a represents an inverted abasic nucleoside. It comprises or consists of:

[0147] In such embodiments, the second strand preferably has the following 5' end motif:

[0148] [ka]

[0149] [In the formula, B represents the nucleoside bases of the first two base nucleosides in the 5'-terminal region of the second strand; T represents a 2'Me ribose modification; V represents O or S (preferably O), R is H or C 1~4 represents alkyl (preferably H), Z comprises 11 to 26 consecutive base nucleosides, preferably 15 to 21 consecutive base nucleosides, more preferably 19 consecutive base nucleosides; More preferably, the following 5'-end motif:

[0150] [ka]

[0151] [In the formula, B represents the nucleoside bases of the first two base nucleosides in the 5'-terminal region of the second strand; T represents a 2'Me ribose modification; Z represents the remaining 19 consecutive nucleosides of the second strand. Includes:

[0152] In a preferred embodiment, the modification pattern of the second (sense) strand of the nucleic acid according to the present invention is i-i-a-Me(s)-Me(s)-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, where (s) is a phosphorothioate internucleoside linkage and i-a represents an inverted abasic nucleoside. It comprises or consists of:

[0153] In such embodiments, the second strand preferably has the following 5' end motif:

[0154] [ka]

[0155] [In the formula, B represents the nucleoside bases of the first two base nucleosides in the 5'-terminal region of the second strand; T represents a 2'Me ribose modification; V represents O or S (preferably O), R is H or C 1~4represents alkyl (preferably H), Z comprises 11 to 26 consecutive base nucleosides, preferably 15 to 21 consecutive base nucleosides, more preferably 19 consecutive base nucleosides; More preferably, the following 5'-end motif:

[0156] [ka]

[0157] [In the formula, B represents the nucleoside bases of the first two base nucleosides in the 5'-terminal region of the second strand; T represents a 2'Me ribose modification; Z represents the remaining 19 consecutive nucleosides of the second strand. Includes:

[0158] Nucleic acid length In one embodiment, i) the first strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides, and / or ii) the second strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0159] Typically, the duplex region of the nucleic acid is between 17 and 30 nucleosides in length, more preferably 19 or 21 nucleosides in length. Similarly, the region of complementarity between the first strand and a portion of the RNA transcribed from the target gene is between 17 and 30 nucleosides in length.

[0160] Nucleic acid modification In certain embodiments, the nucleic acids, eg, RNA, eg, dsiRNA, of the invention do not comprise further modifications, eg, chemical modifications or conjugations known in the art and described herein.

[0161] In other preferred embodiments, the nucleic acids of the invention, eg, RNA, eg, dsiRNA, are further chemically modified to enhance stability or other beneficial characteristics.

[0162] In certain embodiments of the invention, substantially all of the nucleosides are modified.

[0163] Nucleic acids featured in the invention can be synthesized or modified by methods well 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, N.Y., USA, incorporated herein by reference.

[0164] Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleosides in RNA or RNA nucleosides in DNA, inverted linkage, etc.); base modifications, such as replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; or backbone modifications, including modification or replacement of phosphodiester linkages.

[0165] Specific examples of nucleic acids such as siRNA compounds useful in the embodiments described herein include, but are not limited to, RNA containing modified backbone or RNA that does not contain natural internucleoside linkages.Nucleic acids such as RNA with modified backbone include, among others, those that do not have phosphorus atoms in their backbone.For the purpose of this specification and as sometimes referred to in the art, modified nucleic acids such as RNA that do not have phosphorus atoms in their internucleoside backbone can also be considered as oligonucleosides.In some embodiments, modified nucleic acids such as siRNA have phosphorus atoms in their internucleoside backbone.

[0166] Modified nucleic acids, e.g., RNA backbones, include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, their 2'-5' linked analogs, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 5'-3' or 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0167] Modified nucleic acids, such as RNA, can also contain one or more substituted sugar moieties.Nucleic acids, such as siRNAs, such as dsiRNAs, characterized herein can include one of the following at 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl can be substituted or unsubstituted.2'O-methyl and 2'-F are preferred modifications.

[0168] In certain preferred embodiments, the nucleic acid comprises at least one modified nucleoside.

[0169] The nucleic acids of the invention may contain one or more modified nucleosides on the first strand and / or the second strand.

[0170] In some embodiments, substantially all of the nucleosides in the sense strand and all of the nucleosides in the antisense strand comprise a modification.

[0171] In some embodiments, all of the nucleosides in the sense strand and substantially all of the nucleosides in the antisense strand comprise a modification.

[0172] In some embodiments, all of the nucleosides in the sense strand and all of the nucleosides in the antisense strand comprise a modification.

[0173] In one embodiment, at least one of the modified nucleosides is a deoxy-nucleoside, a 3'-terminal deoxy-thymine (dT) nucleoside, a 2'-O-methyl modified nucleoside (also referred to herein as 2'-Me, where Me is methoxy), a 2'-fluoro modified nucleoside, a 2'-deoxy-modified nucleoside, a locked nucleoside, an unlocked nucleoside, a conformationally restricted nucleoside, a constrained ethyl nucleoside, an abasic nucleoside, a 2'-amino-modified nucleoside, a 2'-O-allyl-modified nucleoside, a 2'-C-alkyl In another embodiment, the modified nucleoside is selected from the group consisting of 3'-modified nucleosides, 2'-hydroxyl-modified nucleosides, 2'-methoxyethyl-modified nucleosides, 2'-O-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates, nucleosides containing unnatural bases, tetrahydropyran-modified nucleosides, 1,5-anhydrohexitol-modified nucleosides, cyclohexenyl-modified nucleosides, nucleosides containing phosphorothioate groups, nucleosides containing methylphosphonate groups, nucleosides containing 5'-phosphates, and nucleosides containing 5'-phosphate mimetics. In another embodiment, the modified nucleoside comprises a short sequence of 3'-terminal deoxythymine nucleosides (dT).

[0174] The modification on the nucleoside may preferably be selected from the 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'-hydroxyl, and combinations thereof. In another embodiment, the modification on the nucleoside is a 2'-O-methyl ("2'-Me") or 2'-fluoro modification.

[0175] One preferred modification is a modification at the 2'-OH group of the ribose sugar, optionally selected from a 2'-Me or a 2'-F modification.

[0176] Preferred nucleic acids include one or more nucleosides on the first strand and / or the second strand that are modified to form modified nucleosides, as follows:

[0177] A nucleic acid wherein the modification is at the 2'-OH group of the ribose sugar, optionally selected from a 2'-Me or a 2'-F modification.

[0178] A nucleic acid, wherein each of the first and second strands comprises a 2'-Me and a 2'-F modification.

[0179] A nucleic acid, wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of four or six 2'F modifications.

[0180] A nucleic acid wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5, or 7 2'F modifications.

[0181] A nucleic acid, wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of three 2'F modifications.

[0182] A nucleic acid, wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of five 2'F modifications.

[0183] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 wherein X2, X3, and X4 are selected from 2'Me and 2'F sugar modifications, provided that at least one of X2, X3, and X4 is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications. A nucleic acid comprising a 2' sugar modification pattern of:

[0184] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 [Wherein X2 is a 2'F sugar modification, and X3 and X4 are 2'Me sugar modifications] A nucleic acid comprising a 2' sugar modification pattern of:

[0185] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 [Wherein, X3 is a 2'F sugar modification, and X2 and X4 are 2'Me sugar modifications] A nucleic acid comprising a 2' sugar modification pattern of:

[0186] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 wherein X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications. A nucleic acid comprising a 2' sugar modification pattern of:

[0187] A nucleic acid, wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of 7 2'F modifications.

[0188] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 wherein X2, X3, and X4 are selected from 2'Me and 2'F sugar modifications, provided that at least one of X2, X3, and X4 is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications. A nucleic acid comprising a 2' sugar modification pattern of:

[0189] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 [Wherein X2 is a 2'F sugar modification, and X3 and X4 are 2'Me sugar modifications] A nucleic acid comprising a 2' sugar modification pattern of:

[0190] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 [Wherein, X3 is a 2'F sugar modification, and X2 and X4 are 2'Me sugar modifications] A nucleic acid comprising a 2' sugar modification pattern of:

[0191] The first strand is the following (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 wherein X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications. A nucleic acid comprising a 2' sugar modification pattern of:

[0192] Suitably, a nucleic acid comprising at least one thermally destabilising modification at one or more of positions 1 to 9 of the first strand, counting from position 1 of the first strand, and / or at one or more of the positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilising modification is selected from modified unlocked nucleic acids (UNAs) and glycol nucleic acids (GNAs), preferably glycol nucleic acids, more preferably (S)-glycol nucleic acids.

[0193] A nucleic acid comprising at least one thermodestabilizing modification at position 6 of the first strand, counting from position 1 of the first strand.

[0194] The first strand is the following (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 wherein X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0195] The first strand is the following (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 wherein X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0196] The second strand is the following (5'-3'): (Me)8-(F)3-(Me) 10 A nucleic acid comprising a 2' sugar modification pattern of:

[0197] The second strand is the following (5'-3'): (Me)8-(F)3-(Me) 10 wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of 4 or 6 2'F modifications.

[0198] The second strand is the following (5'-3'): (Me)8-(F)3-(Me) 10 wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5, or 7 2'F modifications.

[0199] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, where X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0200] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): (Me-F)3-(Me)7-F-Me-F-(Me)7 A nucleic acid comprising a 2' sugar modification pattern of:

[0201] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5 A nucleic acid comprising a 2' sugar modification pattern of:

[0202] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3 A nucleic acid comprising a 2' sugar modification pattern of:

[0203] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7, where X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0204] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6 A nucleic acid comprising a 2' sugar modification pattern of:

[0205] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5 A nucleic acid comprising a 2' sugar modification pattern of:

[0206] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 wherein the nucleosides of the first strand are (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3 A nucleic acid comprising a 2' sugar modification pattern of:

[0207] The second strand is the following (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. A nucleic acid comprising a 2' sugar and abasic modification pattern of:

[0208] The second strand is the following (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the first strand comprises a 2' sugar and abasic modification pattern wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of 4 or 6 2'F modifications.

[0209] The second strand is the following (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5, or 7 2'F modifications.

[0210] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, where X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0211] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): (Me-F)3-(Me)7-F-Me-F-(Me)7 A nucleic acid comprising a 2' sugar modification pattern of:

[0212] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5 A nucleic acid comprising a 2' sugar modification pattern of:

[0213] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3 A nucleic acid comprising a 2' sugar modification pattern of:

[0214] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 wherein X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0215] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6 A nucleic acid comprising a 2' sugar modification pattern of:

[0216] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5 A nucleic acid comprising a 2' sugar modification pattern of:

[0217] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside. wherein the nucleosides of the first strand comprise the following (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3 A nucleic acid comprising a 2' sugar modification pattern of:

[0218] In certain embodiments, the nucleic acid, for example, the siRNA agent, further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage.For example, the phosphorothioate or methylphosphonate internucleoside linkage may be located at the 3'-end or terminal region of one strand, i.e., the sense strand or the antisense strand, or may be located at the ends of both strands, i.e., the sense strand and the antisense strand.

[0219] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkages are at the 5'-end or terminal region of one strand, i.e., the sense strand or the antisense strand, or at the termini of both strands, the sense strand and the antisense strand.

[0220] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkages are located at both the 5'- and 3'-end or terminal regions of one strand, i.e., the sense strand or the antisense strand, or at the ends of both strands, the sense strand and the antisense strand.

[0221] Any nucleic acid may contain one or more phosphorothioate (PS) modifications within the nucleic acid, for example, at least two PS internucleoside linkages at the ends of the strand.

[0222] At least one of the oligoribonucleoside chains preferably contains at least two consecutive phosphorothioate modifications in the last three nucleosides of the oligonucleoside.

[0223] Thus, the present invention also relates to the following: a nucleic acid as disclosed herein, comprising phosphorothioate internucleoside linkages between at least two or three consecutive positions, respectively, such as in the 5' and / or 3' terminal and / or near-terminal regions of the second strand, whereby the near-terminal regions are preferably adjacent to the terminal regions in which the one or more abasic nucleosides of the second strand are located.

[0224] A nucleic acid as disclosed herein, comprising a phosphorothioate internucleoside linkage between at least two or three consecutive positions in the 5' and / or 3' terminal region of the first strand, respectively, whereby preferably a terminal position in the 5' and / or 3' terminal region of said first strand is attached to its adjacent position by a phosphorothioate internucleoside linkage.

[0225] The nucleic acid strand may be RNA containing abasic nucleosides at the two termini and phosphorothioate internucleoside linkages between the three adjacent nucleosides.

[0226] In a preferred embodiment, the present invention relates to a nucleic acid in which two phosphorothioate internucleoside linkages are present between three consecutive positions in a region near the 5' end of the second strand, respectively, and a first phosphorothioate internucleoside linkage is present between a first base nucleoside and an adjacent second base nucleoside in the region near the 5' end of the second strand, when read from the 5' end, and a second phosphorothioate internucleoside linkage is present between the second base nucleoside and an adjacent third base nucleoside in the region near the 5' end of the second strand.

[0227] In a more preferred embodiment, the present invention relates to a nucleic acid wherein two phosphorothioate internucleoside linkages are present between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, respectively, whereby the terminal nucleoside in each of the 5'-terminal region and the 3'-terminal region of the first strand is attached to each of the 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage, and each of the 5'- and 3'-adjacent penultimate nucleosides is attached to each of the 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage.

[0228] The second strand is the following (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. A nucleic acid comprising a 2' sugar modification pattern of:

[0229] The second strand is the following (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me)10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the first strand comprises a 2' sugar and abasic modification pattern wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of 4 or 6 2'F modifications.

[0230] The second strand is the following (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the first strand comprises a 2' sugar modification pattern, wherein the modifications are selected from at least 2'Me and 2'F sugar modifications, and wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5, or 7 2'F modifications.

[0231] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)(Me)3-X1-(Me)7-F-Me-F-(Me)5(s)Me(s)Me, where X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0232] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)Me-F-Me-F-(Me)7-F-Me-F-(Me)5(s)Me(s)Me A nucleic acid comprising a 2' sugar modification pattern of:

[0233] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)(Me)3-F-(Me)7-(F-Me)2-F-(Me)3(s)Me(s)Me A nucleic acid comprising a 2' sugar modification pattern of:

[0234] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)(Me)3-F-(Me)7-F-Me-F-(Me)3-F-Me(s)Me(s)Me A nucleic acid comprising a 2' sugar modification pattern of:

[0235] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)5(s)Me(s)Me, where X1 is a thermolabilizing modification. A nucleic acid comprising a 2' sugar modification pattern of:

[0236] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)Me-F-Me-F-Me-(F)2-(Me)4-(F-Me)2-(Me)4(s)Me(s)Me A nucleic acid comprising a 2' sugar modification pattern of:

[0237] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)3(s)Me(s)Me A nucleic acid comprising a 2' sugar modification pattern of:

[0238] 1. A nucleic acid comprising: a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene; and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region at least 17 nucleosides in length, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage. wherein the nucleosides of the first strand comprise the following (5'-3'): Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-Me(s)Me(s)Me A nucleic acid comprising a 2' sugar modification pattern of:

[0239] Position 1 of the first or second strand is the nucleoside that is closest to the end of the nucleic acid (disregarding any abasic nucleosides) and that is connected to the adjacent nucleoside (at position 2) via a 3' to 5' internal bond, reading away from that end of the molecule, with respect to the bond between the sugar moieties of the backbone.

[0240] Thus, "position 1 of the sense strand" can be considered to be the 5'-most nucleoside (not including the abasic nucleoside) at the conventional 5'-end of the sense strand. Typically, the nucleoside at this position 1 of the sense strand is equivalent to the 5' nucleoside of a selected target nucleic acid sequence; more commonly, the sense strand has nucleosides equivalent to those of the target nucleic acid sequence starting at this position 1 of the sense strand, while also allowing for mismatches between the sequences.

[0241] As used herein, "position 1 of the antisense strand" refers to the 5'-most nucleoside (not including the abasic nucleoside) at the conventional 5'-end of the antisense strand. As described above, there is a complementary region between the sense strand and the antisense strand, and thus the antisense strand also has a complementary region to the target nucleic acid sequence, as described above.

[0242] A preferred nucleic acid is a double-stranded RNA comprising two adjacent abasic nucleosides at the 5'-end of the second strand and a ligand moiety comprising one or more GalNAc ligand moieties at the opposite 3'-end of the second strand. More preferably, the nucleic acid may also comprise phosphorothioate linkages between nucleotides 1-2 and 2-3 of the second strand, reading from position 1 of the second strand.

[0243] Preferred modifications are: Modification pattern 1: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me-F-Me-Me-Me-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, where X1 is a heat destabilizing modification; Or modification pattern 2: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-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 modification pattern 3: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me, or modification pattern 4: 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-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-Me, 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-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me [where X1 is a thermally destabilizing modification], or modification pattern 6: 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-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, or modification pattern 7: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me, or modification pattern 8: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-Me.

[0244] Particularly preferred modifications are: Modification pattern 1: Second strand (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where X1 is a heat destabilizing modification; Or modification pattern 2: Second strand (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-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 modification pattern 3: Second strand (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me, Or modification pattern 4: 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-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me, 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-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me [where X1 is a thermally destabilizing modification], Or modification pattern 6: 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-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, Or modification pattern 7: 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-F-F-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me, Or modification pattern 8: 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-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me where (s) is a phosphorothioate internucleoside linkage.

[0245] Conjugation Another modification of the nucleic acids, e.g., RNA, e.g., siRNA, of the invention includes linking the nucleic acid, e.g., siRNA, to one or more ligand moieties, e.g., to enhance the activity, cellular distribution, or cellular uptake, e.g., into cells, of the nucleic acid, e.g., siRNA.

[0246] In some embodiments, the described ligand moieties may be attached to a nucleic acid, e.g., an siRNA oligonucleoside, via a linker, which may be cleavable or non-cleavable. The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound, e.g., covalently bonds two parts of a compound.

[0247] The ligand may be attached to the 3' or 5' end of the sense strand.

[0248] The ligand is preferably conjugated to the 3' end of the sense strand of the nucleic acid, eg, the siRNA agent.

[0249] Thus, in a further aspect, the present invention relates to a conjugate for inhibiting expression of a target gene in a cell, said conjugate comprising a nucleic acid moiety and one or more ligand moieties, said nucleic acid moiety comprising a nucleic acid disclosed herein.

[0250] In one embodiment, the second strand of the nucleic acid is conjugated, directly or indirectly (e.g., via a linker), to one or more ligand moieties, which are typically present in a terminal region of the second strand, preferably its 3'-terminal region.

[0251] In certain embodiments, the ligand moiety comprises a GalNAc or GalNAc derivative attached to a nucleic acid, eg, a dsiRNA, via a linker.

[0252] Thus, the present invention provides a compound comprising a ligand moiety: i) one or more GalNAc ligands, and / or ii) one or more GalNAc ligand derivatives, and / or iii) one or more GalNAc ligands conjugated to said nucleic acid via a linker; The present invention relates to a conjugate comprising:

[0253] 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 at its 3' terminal region.

[0254] GalNAc ligands are well known in the art and are described, inter alia, in EP3775207A1.

[0255] In some embodiments, the GalNAc ligand is included in any one of the linkers shown in Figures 1-4 or 5 (Formula XI), and the "oligonucleotide" can be any nucleic acid disclosed herein. Thus, an "oligonucleotide" can include linkages other than phosphodiester linkages, for example, one or more phosphorothioate linkages. Preferably, nucleic acids according to the invention are double-stranded oligonucleosides as defined herein, and the linker is conjugated to the second strand, more preferably to the 3'-terminal region of the second strand, via a phosphodiester bond.

[0256] In some embodiments, the GalNAc ligand is included in the linker shown in Figure 3, and the "oligonucleotide" can be any nucleic acid disclosed herein. Thus, an "oligonucleotide" can include a linkage other than a phosphodiester linkage, for example, one or more phosphorothioate linkages. Preferably, a nucleic acid according to the present invention is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand, more preferably to the 3'-terminal region of the second strand, via a phosphodiester bond.

[0257] In some embodiments, the GalNAc ligand is included in the linker shown in Figure 5 (Formula XI), and the "oligonucleotide" can be any nucleic acid disclosed herein. Thus, the "oligonucleotide" can include a bond other than a phosphodiester bond, for example, one or more phosphorothioate bonds. Preferably, the nucleic acid according to the present invention is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand, more preferably to the 3'-terminal region of the second strand, via a phosphodiester bond.

[0258] In some embodiments, the GalNAc ligand is included in any one of the linkers shown in Figures 1-4 or 5 (Formula XI), and "oligonucleotide" refers to a nucleic acid according to the invention, which nucleic acid according to the invention has the following modification pattern (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me wherein (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside. and preferably the linker is conjugated to the 3'-terminal region of the second strand via a phosphodiester bond.

[0259] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 3, and "oligonucleotide" refers to a nucleic acid according to the invention, which has the following modification pattern (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me wherein (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside. and preferably the linker is conjugated to the 3'-terminal region of the second strand via a phosphodiester bond.

[0260] In some embodiments, the GalNAc ligand is included in the linker shown in Figure 5 (Formula XI), and "oligonucleotide" refers to a nucleic acid according to the invention, which has the following modification pattern (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me wherein (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside. and preferably the linker is conjugated to the 3'-terminal region of the second strand via a phosphodiester bond.

[0261] In some embodiments, the GalNAc ligand is included in any one of the linkers shown in Figures 1-4 or 5 (Formula XI), and "oligonucleotide" refers to a nucleic acid according to the invention, which nucleic acid according to the invention has the following modification pattern (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me wherein (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside. and a modified second strand having the following structure:

[0262] [ka]

[0263] [In the formula, T represents a 2'Me ribose modification; B represents the nucleoside bases of the first two base nucleosides in the 5'-terminal region of the second strand; Z represents the remaining 19 consecutive nucleosides of the second strand. It has.

[0264] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 3, and "oligonucleotide" refers to a nucleic acid according to the invention, which has the following modification pattern (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me wherein (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside. and a modified second strand having the following structure:

[0265] [ka]

[0266] [In the formula, T represents a 2'Me ribose modification; B represents the nucleoside bases of the first two base nucleosides in the 5'-terminal region of the second strand; Z represents the remaining 19 consecutive nucleosides of the second strand. It has.

[0267] In some embodiments, the GalNAc ligand is included in the linker shown in Figure 5 (Formula XI), and "oligonucleotide" refers to a nucleic acid according to the invention, which has the following modification pattern (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me wherein (s) is a phosphorothioate internucleoside linkage and ia represents an inverted abasic nucleoside. and a modified second strand having the following structure:

[0268] [ka]

[0269] [In the formula, T represents a 2'Me ribose modification; B represents the nucleoside bases of the first two base nucleosides in the 5'-terminal region of the second strand; Z represents the remaining 19 consecutive nucleosides of the second strand. It has.

[0270] Vectors and cells In one aspect, the invention provides a cell containing a nucleic acid described herein, eg, an inhibitory RNA [RNAi].

[0271] In one aspect, the invention provides a cell comprising a vector described herein.

[0272] Pharmaceutically acceptable compositions In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of a target gene, the pharmaceutical composition comprising a nucleic acid disclosed herein.

[0273] A pharmaceutically acceptable composition may include an excipient and / or carrier.

[0274] Some examples of materials that can serve 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 carboxymethylcellulose, ethylcellulose, 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 wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0275] Typical pharmaceutical carriers include, but are not limited to, binders (such as, for example, pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (such as, for example, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate), lubricants (such as, for example, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate), disintegrants (such as, for example, starch, sodium starch glycolate), and wetting agents (such as, for example, sodium lauryl sulfate).

[0276] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0277] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of nucleic acids in liquid or solid oil bases. Solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not deleteriously react with nucleic acids can be used.

[0278] In one embodiment, the nucleic acid or composition is administered in an unbuffered solution. In certain embodiments, the unbuffered solution is saline or water. In other embodiments, the nucleic acid, for example, the siRNA agent, is administered in a buffered solution. In such embodiments, the buffered solution may contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. For example, the buffered solution may be phosphate-buffered saline (PBS).

[0279] Dosage The pharmaceutical compositions of the present invention can be administered at a dosage sufficient to inhibit gene expression. Generally, suitable doses of the nucleic acids, e.g., siRNA, of the present invention range from about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally from about 1 to 50 mg per kilogram of recipient body weight per day. Typically, suitable doses of the nucleic acids, e.g., siRNA, of the present invention range from about 0.1 mg / kg to about 5.0 mg / kg, e.g., about 0.3 mg / kg and about 3.0 mg / kg.

[0280] A repeat dosing regimen can include administering a therapeutic amount of a nucleic acid, e.g., an siRNA, on a regular basis, such as every other day or once a year. In certain embodiments, the nucleic acid, e.g., an siRNA, is administered about once a month to about once a quarter (i.e., about once every three months).

[0281] In various embodiments, the nucleic acid, e.g., siRNA agent, is administered at 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., siRNA agent, is administered at a dose of about 10 mg / kg to about 30 mg / kg. In certain embodiments, the nucleic acid, e.g., siRNA agent, is administered at 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., agent, is administered at a dose of about 0.1 mg / kg to about 5.0 mg / kg about once per week, once per month, once per two months, or once per quarter (i.e., once every three months). In certain embodiments, the nucleic acid, e.g., siRNA agent, is administered to the subject once per week. In certain embodiments, the nucleic acid, e.g., siRNA agent, is administered to the subject once per month. In certain embodiments, the nucleic acid, eg, siRNA agent, is administered quarterly (ie, every three months).

[0282] After the initial treatment regimen, treatment can be administered less frequently, for example, weekly or biweekly for three months, followed by monthly administration for six months or a year, or longer.

[0283] The pharmaceutical composition can be administered once a day, or as two, three or more partial doses at appropriate intervals throughout the day, or even by continuous infusion or delivery via controlled release formulation.In this case, the nucleic acid, such as siRNA, contained in each partial dose must be correspondingly smaller to achieve the total daily dosage.The dosage unit can also be configured for delivery over several days, for example, by using a conventional sustained release formulation that provides sustained release of nucleic acid, such as siRNA, over several days.Sustained release formulations are well known in the art and are particularly useful for the delivery of drugs at specific sites, such as those that can be used in the drugs of the present invention.In this embodiment, the dosage unit contains a corresponding number of daily doses.

[0284] In other embodiments, a single dose of the pharmaceutical composition may be long-lasting, such that subsequent doses are administered at intervals not exceeding 3, 4, or 5 days, or not exceeding 1, 2, 3, or 4 weeks. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In other embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered every other month. In certain embodiments, the siRNA is administered about once a month to about once a quarter (i.e., about once every 3 months), or even every 6 or 12 months.

[0285] Estimation of effective dosages and in vivo half-lives for particular nucleic acids encompassed by the invention, e.g., siRNAs, can be made using conventional methods or based on in vivo studies using appropriate animal models as known in the art.

[0286] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary, e.g., by inhalation or insufflation of a powder or aerosol, e.g., 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 implanted device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular, administration. In certain preferred embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

[0287] In one embodiment, the nucleic acid, eg, the drug, is administered subcutaneously to the subject.

[0288] Nucleic acids, such as siRNA, can be delivered in a manner that targets specific tissues (eg, particularly in liver cells).

[0289] Methods for inhibiting target gene expression The present invention also provides a method for inhibiting the expression of target gene in cell.Method comprises contacting cell with the nucleic acid of the present invention, for example, siRNA agent, for example, double-stranded siRNA agent, in an amount effective for inhibiting the expression of target gene in cell, thereby inhibiting the expression of target gene in cell.It should be noted that the nucleic acid for " inhibiting the expression of target gene " is preferably the nucleic acid that can inhibit target gene expression, as described herein below.

[0290] Contacting a cell with a nucleic acid, e.g., an siRNA, e.g., a double-stranded siRNA agent, can be performed in vitro or in vivo. Contacting a cell with a nucleic acid in vivo includes, for example, contacting a cell or a group of cells in a subject, e.g., a human subject, with a nucleic acid, e.g., an siRNA. A combination of in vitro and in vivo cell contacting methods is also possible. Contacting a cell can be direct or indirect, as discussed above. Furthermore, contacting a cell can be achieved via a targeting ligand moiety, including any ligand moiety described herein or known in the art. In a preferred embodiment, the targeting ligand moiety is a carbohydrate moiety, e.g., a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to a target site.

[0291] The term "inhibiting," as used herein, is used interchangeably with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes any level of inhibition.

[0292] In some embodiments of the methods of the invention, 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 to below the detection level of the assay, preferably as determined by qPCR as described herein and / or when the siRNA is introduced into the target cells by transfection. In certain embodiments, the methods include clinically relevant inhibition of expression of the target gene, as demonstrated, for example, by clinically relevant results after treatment of the subject with an agent that reduces expression of the gene.

[0293] In some embodiments, when transfected into a cell, the nucleic acids of the invention preferably inhibit expression of a target gene with an IC50 value of less than 2500 pM, 2400 pM, 2300 pM, 2200 pM, 2100 pM, 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, as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0294] Inhibition of the target gene can be quantified by the following method: Huh7 cells (a human hepatocyte-derived cell line obtained from the JCRB Cell Bank) can be maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO atmosphere. Cells can then be transfected with siRNA duplexes targeting mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 115), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 114)) using 10x3-fold serial dilutions spanning a final duplex concentration range of 20 nM to 1 pM. Transfections can be performed by adding 9.7 μL of Opti-MEM (ThermoFisher) plus 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. The cells can be incubated at 37°C / 5% CO2 for 24 hours, after which total RNA purification can be performed using the RNeasy 96 Kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in a single experiment.

[0295] cDNA synthesis can be performed using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the FastStart Universal Probe Master Kit (Roche) with primers specific for the target gene and human GAPDH (Hs02786624_g1).

[0296] qPCR is performed in duplicate on cDNA from each well, and the mean cycle threshold (Ct) can be calculated. Percent maximal inhibition of target gene expression and IC50 values can be calculated using a four-parameter (variable slope) model using GraphPad Prism 9.

[0297] Alternatively or additionally, inhibition of expression of a target gene may be characterized by a decrease in the average relative expression of the target gene.

[0298] In some embodiments, when cells are transfected with 0.1 nM of a nucleic acid of the invention, the average relative expression of the target gene is preferably less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4, as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0299] In some embodiments, when cells are transfected with 5 nM of a nucleic acid of the invention, 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 as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0300] The average relative expression of a target gene can be quantified by the following method: Huh7 cells (a human hepatocyte-derived cell line obtained from the JCRB Cell Bank) can be maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO atmosphere. Cells can be transfected with siRNA duplexes targeting mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 115), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 114)) at final duplex concentrations of 5 nM and 0.1 nM. Transfection can be performed by adding 9.7 μL of Opti-MEM (ThermoFisher) plus 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. The cells can be incubated at 37°C / 5% CO2 for 24 hours, after which total RNA purification can be performed using the RNeasy 96 Kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in two independent experiments.

[0301] cDNA synthesis can be performed using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the FastStart Universal Probe Master Kit (Roche) with primers specific for the target gene and human GAPDH (Hs02786624_g1).

[0302] qPCR can be performed in duplicate on cDNA from each well and the average Ct can be calculated. Relative target gene expression can be calculated from the average Ct values, normalized to GAPDH, relative to untreated cells using the comparative Ct (ΔΔCt) method.

[0303] Inhibition of target gene expression may be manifested by a reduction in the amount of target gene mRNA compared to a suitable control.

[0304] In other embodiments, inhibition of target gene expression can be assessed with respect to a parameter functionally related to gene expression, such as a reduction in protein expression or signaling pathways. Examples of target genes exemplified herein are HCII, ZPI, and B4GALT1.

[0305] Methods for treating or preventing diseases associated with target gene expression The present invention also provides a method for using the nucleic acid of the present invention, for example, siRNA, or a composition containing the nucleic acid of the present invention, for example, siRNA, to reduce or inhibit target gene expression in cells.The method includes contacting cells with the nucleic acid of the present invention, for example, dsiRNA, and maintaining the cells for a sufficient time to obtain the degradation of target mRNA transcripts, thereby inhibiting the expression of target gene in cells.The reduction of gene expression can be evaluated by any method known in the art.

[0306] In the methods of the present invention, the cells may be contacted in vitro or in vivo, ie, the cells may be within a subject.

[0307] Cells suitable for treatment using the methods of the present invention may be any cells that express a gene of interest associated with a disease involving impaired hemostasis, such as hemophilia, particularly when the gene of interest is ZPI or HCII.

[0308] Alternatively, cells suitable for treatment using the methods of the present invention may be any cell that expresses a gene of interest associated with diabetes or cardiovascular disease, particularly where the gene of interest is B4GALT1.

[0309] The in vivo methods of the present invention may include administering to a subject a composition containing a nucleic acid, e.g., an siRNA, of the present invention, wherein the nucleic acid, e.g., the siRNA, comprises a nucleoside sequence that is complementary to at least a portion of an RNA transcript of a target gene in the mammal to be treated.

[0310] The present invention further provides a method for treating a subject in need thereof. The treatment method of the present invention comprises administering a nucleic acid, such as an siRNA, of the present invention to a subject, for example, a subject who would benefit from reduced or inhibited expression of a target gene, in a therapeutically effective amount, for example, in a pharmaceutical composition comprising a nucleic acid, such as an siRNA against the target gene, or a nucleic acid that targets the gene.

[0311] The disease to be treated may relate to a disorder of hemostasis, such as a disease relating to a disorder of hemostasis, such as hemophilia, for example when the target gene is HCII or ZPI as disclosed herein.

[0312] Hemophilia, or hemophilia, is a genetic disorder, mostly inherited, that impairs the body's ability to produce blood clots, a process required to stop bleeding. This results in subjects bleeding for longer periods after injury, easy bruising, and an increased risk of bleeding into joints or the brain. Subjects with mild disease may only experience symptoms after an accident or during surgery. Bleeding into joints, also known as hemarthrosis, can cause permanent damage, while bleeding in the brain can cause long-term headaches, seizures, or a decreased level of consciousness.

[0313] There are two main types of hemophilia: hemophilia A, which is caused by low levels of clotting factor VIII, and hemophilia B, which is caused by low levels of clotting factor IX. They are typically inherited from parents through the X chromosome, which carries a nonfunctional gene. Rarely, new mutations can occur during early development, or hemophilia can develop later in life due to antibodies formed against clotting factors. Other types include hemophilia C, which is caused by low levels of factor XI; von Willebrand disease, which is caused by low levels of a substance called von Willebrand factor; and parahemophilia, which is caused by low levels of factor V. Hemophilia A, B, and C prevent the intrinsic pathway from functioning properly; this clotting pathway is necessary when the endothelium of blood vessels is damaged. Acquired hemophilia is associated with cancer, autoimmune disorders, and pregnancy. Diagnosis is by testing blood for its clotting ability and clotting factor levels.

[0314] In certain embodiments, nucleic acids of the invention, particularly nucleic acids that inhibit expression of ZPI or HCII, are suitable for the treatment of hemophilia A, B, and / or C. In certain embodiments, nucleic acids of the invention, particularly nucleic acids that inhibit expression of ZPI or HCII, are suitable for the treatment of hemophilia A and / or B. In certain embodiments, nucleic acids of the invention, particularly nucleic acids that inhibit expression of ZPI or HCII, are suitable for the treatment of acquired hemophilia. In certain embodiments, nucleic acids of the invention, particularly nucleic acids that inhibit expression of ZPI or HCII, are suitable for the treatment of Willebrand's disease. In certain embodiments, nucleic acids of the invention, particularly nucleic acids that inhibit expression of ZPI or HCII, are suitable for the treatment of parahemophilia.

[0315] Without wishing to be bound by theory, treatment with the nucleic acids of the present invention may result in an increase in clotting factor levels, such that bleeding may be reduced or prevented. Thus, in a preferred embodiment, treatment with the nucleic acids of the present invention, particularly nucleic acids that inhibit the expression of ZPI or HCII, may reduce or prevent bleeding episodes in a subject suffering from hemophilia. In another preferred embodiment, treatment with the nucleic acids of the present invention, particularly nucleic acids that inhibit the expression of ZPI or HCII, may reduce or prevent bleeding into the joints of a subject suffering from hemophilia. In certain embodiments, treatment with the nucleic acids of the present invention, particularly nucleic acids that inhibit the expression of ZPI or HCII, may reduce or prevent bleeding into the muscles or brain of a subject suffering from hemophilia.

[0316] The disease to be treated can be diabetes, particularly when the target gene is B4GALT1 as disclosed herein.

[0317] According to the present invention, the term "diabetes" as used herein refers to a group of metabolic diseases in which a subject suffers from hyperglycemia due to the body not producing enough insulin or because cells do not respond to the insulin produced. There are three main types of diabetes: (1) Type 1 diabetes (T1D): resulting from the body's inability to produce insulin, requiring the individual to inject insulin (also known as insulin-dependent diabetes mellitus, abbreviated IDDM, and juvenile diabetes). (2) Type 2 diabetes (T2D): resulting from insulin resistance, a condition in which cells are unable to properly use insulin, sometimes combined with absolute insulin deficiency (previously known as non-insulin-dependent diabetes mellitus, abbreviated NIDDM, and adult-onset diabetes). (3) Gestational diabetes (GD): when a pregnant woman who has never had diabetes before has high blood glucose levels during pregnancy. This may precede the onset of T2D.

[0318] In one particular embodiment, the nucleic acids according to the invention, in particular the nucleic acids that inhibit the expression of B4GALT1, or the pharmaceutical compositions comprising said nucleic acids, are used for the treatment of diabetes, preferably type 2 diabetes (T2D).

[0319] The disease to be treated can be a cardiovascular disease, particularly when the target gene is B4GALT1 as disclosed herein.

[0320] The term "cardiovascular disease," as used herein, refers to any condition, disorder, or pathology associated with, resulting from, or causing a structural or functional abnormality of the heart or the blood vessels supplying the heart that impairs its normal function. Cardiovascular disease 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.

[0321] In certain embodiments, the nucleic acid of the present invention, particularly the nucleic acid that inhibits the expression of B4GALT1, or a pharmaceutical composition containing the nucleic acid, is used to treat cardiovascular disease, preferably coronary artery disease. The nucleic acid of the present invention, such as siRNA, may be administered as a "free" nucleic acid or "free" siRNA administered in the absence of a pharmaceutical composition. The naked nucleic acid may be in a suitable buffer solution. The buffer solution may contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmolality of the buffer solution can be adjusted to be suitable for administration to a subject.

[0322] Alternatively, the nucleic acids of the invention, eg, siRNA, can be administered as pharmaceutical compositions, eg, dsiRNA liposome formulations.

[0323] In one embodiment, the method includes administering a composition featured herein such that expression of the target gene is reduced, e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, or about 36 hours. In one embodiment, expression of the target gene is reduced for an extended period of time, e.g., at least about 2, 3, 4 days or more, e.g., for about 1 week, 2 weeks, 3 weeks, or 4 weeks or more, e.g., for about 1 month, 2 months, or 3 months.

[0324] A subject can be administered a therapeutic amount of a nucleic acid, e.g., an siRNA, such as about 0.01 mg / kg to about 200 mg / kg, to treat a disease associated with impaired hemostasis, e.g., a disease associated with impaired hemostasis, such as hemophilia, or to treat diabetes or cardiovascular disease.

[0325] Nucleic acids, such as siRNA, can be administered by intravenous infusion periodically over a period of time. In certain embodiments, after the initial treatment regimen, treatment can be administered less frequently. For example, administration of siRNA can reduce the gene product level of the target gene in the 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 to below the detection level of the assay method used. In certain embodiments, administration results in clinical stabilization or preferably clinically relevant reduction of at least one sign or symptom of target gene-related disorder.

[0326] Alternatively, nucleic acid, for example, siRNA, can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver a desired daily dose of nucleic acid, for example, siRNA, to a subject. Injection can be repeated over a period of time. Administration can be repeated periodically. In certain embodiments, after an initial treatment regimen, treatment can be administered less frequently. Repeated administration regimens can include administering a therapeutic amount of nucleic acid on a regular basis, such as every other day or once a year. In certain embodiments, nucleic acid is administered about once a month to about once a quarter (i.e., about once every three months).

[0327] In one aspect, the present invention can be applied to compounds, methods, compositions, or uses in the following sentences numbered 1-101, and reference to any formula in sentences 1-101 refers only to the formula defined in sentences 1-101. These formulas are reproduced in Figure 5. Specifically, the oligonucleoside moiety represented by Z in any of the following sentences can comprise a nucleic acid for inhibiting expression of ZPI, HCII, or B4GALT1 as defined herein below.

[0328] 1. The following structure:

[0329] [ka]

[0330] [In the formula, R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl, and ethyl; R2 is hydrogen, hydroxy, -OC 1~3 Alkyl, -C(=O)OC 1~3 selected from the group consisting of alkyl, halo, and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen, and sulfur; m is an integer from 1 to 6; n is an integer from 1 to 10, q, r, s, t, and v are independently integers of 0 to 4, with the proviso that (i) q and r cannot both be 0 at the same time, and (ii) s, t, and v cannot all be 0 simultaneously; Z is an oligonucleoside moiety. A compound comprising:

[0331] 2. The compound according to the first sentence, wherein each occurrence of R1 is hydrogen.

[0332] 3. The compound according to the first sentence, wherein R1 is methyl.

[0333] 4. The compound according to the first sentence, wherein R1 is ethyl.

[0334] 5. A compound according to any one of the first to fourth sentences, wherein R2 is hydroxy.

[0335] 6. The compound according to any one of the first to fourth sentences, wherein R2 is halo.

[0336] 7. The compound according to sentence 6, wherein R2 is fluoro.

[0337] 8. The compound according to sentence 6, wherein R2 is chloro.

[0338] 9. The compound according to sentence 6, wherein R2 is bromo.

[0339] 10. The compound according to sentence 6, wherein R2 is iodo.

[0340] 11. The compound according to sentence 6, wherein R2 is nitro.

[0341] 12. The compound according to any one of the first to eleventh sentences, wherein X1 is methylene.

[0342] 13. The compound according to any one of the first to eleventh sentences, wherein X1 is oxygen.

[0343] 14. The compound according to any one of the first to eleventh sentences, wherein X1 is sulfur.

[0344] 15. The compound according to any one of sentences 1 to 14, wherein X2 is methylene.

[0345] 16. The compound according to any one of the first to fifteenth sentences, wherein X2 is oxygen.

[0346] 17. The compound according to any one of sentences 1 to 16, wherein X2 is sulfur.

[0347] 18. A compound according to any one of the first to seventeenth sentences, wherein m=3.

[0348] 19. A compound according to any one of the first to eighteenth sentences, wherein n=6.

[0349] 20. X1 is oxygen and X2 is methylene, preferably q=1, r=2, s=1, t=1, v=1, Compounds according to sentences 13 and 15.

[0350] 21. Both X1 and X2 are methylene, preferably q=1, r=3, s=1, t=1, v=1, Compounds according to sentences 12 and 15.

[0351] 22. Z is

[0352] [ka]

[0353] [In the formula, Z1, Z2, Z3, and Z4 are independently oxygen or sulfur at each occurrence; one of the bonds between P and Z2 and between P and Z3 is a single bond, and the other is a double bond. The compound according to any one of the first to twenty-first sentences,

[0354] 23. A compound according to sentence 22, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0355] 24. The compound of sentence 23, wherein the RNA compound comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, and each of the first and second strands having a 5' and a 3' end.

[0356] 25. The compound of sentence 24, wherein the RNA compound is attached at the 5' end of its second strand to an adjacent phosphate.

[0357] 26. The compound according to sentence 24, wherein the RNA compound is attached at the 3' end of its second strand to adjacent phosphates.

[0358] 27. Compound of formula (II):

[0359] [ka]

[0360] 28. Compounds of formula (III):

[0361] [ka]

[0362] 29. A compound according to sentence 27 or 28, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex being attached to adjacent phosphates at the 5' end of the second strand.

[0363] 30. A composition comprising a compound of formula (II) as defined in sentence 27 and a compound of formula (III) as defined in sentence 28, optionally dependent on sentence 29.

[0364] 31. A composition according to sentence 30, wherein the compound of formula (III) as defined in sentence 28 is present in an amount ranging from 10 to 15% by weight of the composition.

[0365] 32. Compound of formula (IV):

[0366] [ka]

[0367] 33. A compound of formula (V):

[0368] [ka]

[0369] 34. A compound according to sentence 32 or 33, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex being attached to adjacent phosphates at the 3' end of the second strand.

[0370] 35. A composition comprising a compound of formula (IV) as defined in sentence 32 and a compound of formula (V) as defined in sentence 33, optionally dependent on sentence 34.

[0371] 36. A composition according to sentence 35, wherein the compound of formula (V) as defined in sentence 33 is present in an amount ranging from 10 to 15% by weight of the composition.

[0372] 37. A compound as defined in any one of sentences 1 to 29 or 32 to 34, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2' position, preferably multiple riboses modified at the 2' position.

[0373] 38. The compound according to sentence 37, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0374] 39. A compound according to any one of sentences 1 to 29, or sentences 32 to 34, or sentences 37 to 38, wherein the oligonucleoside further comprises one or more degradation-protecting moieties at one or more termini.

[0375] 40. The compound of sentence 39, wherein the one or more degradation protecting moieties are not present at the termini of the oligonucleoside chain bearing the ligand moiety, and / or the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleosides are present at the distal termini of the chain bearing the ligand moiety.

[0376] 41. A compound described in any one of sentences 1 to 29, sentences 32 to 34, or sentences 37 to 40, wherein the ligand portion represented by formula (I) in sentence 1 includes one or more ligands.

[0377] 42. The compound according to sentence 41, wherein the ligand moiety shown in formula (I) in the first sentence comprises one or more carbohydrate ligands.

[0378] 43. The compound according to sentence 42, wherein the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide.

[0379] 44. The compound described in sentence 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.

[0380] 45. The compound described in sentence 44, wherein the one or more carbohydrates contain one or more N-acetyl-galactosamine moieties.

[0381] 46. A compound according to sentence 45, containing two or three N-acetylgalactosamine moieties.

[0382] 47. A compound according to any one of sentences 41 to 46, wherein the one or more ligands are attached in a linear or branched configuration.

[0383] 48. The compound according to sentence 47, wherein the one or more ligands are attached in a biantennary or triantennary branched configuration.

[0384] 49. The moiety shown in formula (I) in the first sentence:

[0385] [ka]

[0386] is any of formula (VIa), (VIb) or (VIc), preferably formula (VIa):

[0387] [ka]

[0388] [In the formula, A I is hydrogen or a suitable hydroxy protecting group; a is an integer of 2 or 3, b is an integer from 2 to 5; or

[0389] [ka]

[0390] [In the formula, A I is hydrogen or a suitable hydroxy protecting group; a is an integer of 2 or 3, c and d are independently integers from 1 to 6; or

[0391] [ka]

[0392] [In the formula, A I is hydrogen or a suitable hydroxy protecting group; a is an integer of 2 or 3, e is an integer between 2 and 10. The compound according to any one of the 46th to 48th sentences,

[0393] 50. The moiety shown in formula (I) in the first sentence:

[0394] [ka]

[0395] is represented by formula (VII):

[0396] [ka]

[0397] [In the formula, A I is hydrogen, a is an integer of 2 or 3. The compound according to any one of the 46th to 48th sentences,

[0398] 51. A compound according to sentence 49 or 50, wherein a=2.

[0399] 52. A compound according to sentence 49 or 50, wherein a=3.

[0400] 53. The compound according to sentence 49, wherein b=3.

[0401] 54. Compound of formula (VIII):

[0402] [ka]

[0403] 55. A compound of formula (IX):

[0404] [ka]

[0405] 56. A compound according to sentence 54 or 55, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex being attached to adjacent phosphates at the 5' end of the second strand.

[0406] 57. A composition comprising a compound of formula (VIII) as defined in sentence 54 and a compound of formula (IX) as defined in sentence 55, optionally dependent on sentence 56.

[0407] 58. A composition according to sentence 57, wherein the compound of formula (IX) as defined in sentence 55 is present in an amount ranging from 10 to 15% by weight of the composition.

[0408] 59. A compound of formula (X):

[0409] [ka]

[0410] 60. A compound of formula (XI):

[0411] [ka]

[0412] 61. A compound according to sentences 59 or 60, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex being attached to adjacent phosphates at the 3' end of the second strand.

[0413] 62. A composition comprising a compound of formula (X) as defined in sentence 59 and a compound of formula (XI) as defined in sentence 60, optionally dependent on sentence 61.

[0414] 63. A composition according to sentence 62, wherein the compound of formula (XI) as defined in sentence 60 is present in an amount ranging from 10 to 15% by weight of the composition.

[0415] 64. A compound as defined in any one of sentences 54 to 63, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2' position, preferably multiple riboses modified at the 2' position.

[0416] 65. The compound according to sentence 64, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0417] 66. A compound according to any one of sentences 54 to 65, wherein the oligonucleoside further comprises one or more degradation-protecting moieties at one or more termini.

[0418] 67. The compound of sentence 66, wherein the one or more degradation protecting moieties are not present at the termini of the oligonucleoside chain bearing the ligand moiety, and / or the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleosides are present at the distal termini of the chain bearing the ligand moiety, as shown in any of formulas (VIII), (IX), (X), or (XI) in any one of sentences 54, 55, 59, or 60.

[0419] 68. Compounds of formula (XII) and (XIII):

[0420] [ka]

[0421] [In the formula, R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl, and ethyl; R2 is hydrogen, hydroxy, -OC 1~3 Alkyl, -C(=O)OC 1~3 selected from the group consisting of alkyl, halo, and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen, and sulfur; m is an integer from 1 to 6; n is an integer from 1 to 10, q, r, s, t, and v are independently integers of 0 to 4, with the proviso that (i) q and r cannot both be 0 at the same time, and (ii) s, t, and v cannot all be 0 simultaneously; Z is an oligonucleoside moiety. and, if appropriate, deprotecting the ligand and / or annealing the second strand of the oligonucleoside moiety,

[0422] 69. The compound of formula (XII) is a compound of formula (XIV) and (XV):

[0423] [ka]

[0424] [In the formula, R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl, and ethyl; R2 is hydrogen, hydroxy, -OC 1~3 Alkyl, -C(=O)OC 1~3 selected from the group consisting of alkyl, halo, and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen, and sulfur; q, r, s, t, and v are independently integers of 0 to 4, with the proviso that (i) q and r cannot both be 0 at the same time, and (ii) s, t, and v cannot all be 0 simultaneously; Z is an oligonucleoside moiety. 68. The method according to claim 68, wherein the compound is prepared by reacting

[0425] 70. The compound of formula (XII) is a compound of formula (XIIa):

[0426] [ka]

[0427] and the compound of formula (XIII) is of formula (XIIIa):

[0428] [ka]

[0429] The method of any one of sentences 20, 25, 27, 29, 54, and 56 for preparing a compound and / or a composition of any one of sentences 30, 31, 57, and 58, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, and the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex is attached to adjacent phosphates at the 5' end of the second strand.

[0430] 71. The compound of formula (XII) is a compound of formula (XIIb):

[0431] [ka]

[0432] and the compound of formula (XIII) is of formula (XIIIa):

[0433] [ka]

[0434] The method of any one of sentences 20, 25, 28, 29, 55, and 56 for preparing a compound and / or a composition of any one of sentences 30, 31, 57, and 58, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, and the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex is attached to adjacent phosphates at the 5' end of the second strand.

[0435] 72. The compound of formula (XII) is a compound of formula (XIIc):

[0436] [ka]

[0437] and the compound of formula (XIII) is of formula (XIIIa):

[0438] [ka]

[0439] 68. The method of claim 68 for preparing a compound according to any one of sentences 21, 26, 32, 34, 59, and 61, and / or a composition according to any one of sentences 35, 36, 62, and 63, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, and the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex is attached to adjacent phosphates at the 3' end of the second strand.

[0440] 73. The compound of formula (XII) is a compound of formula (XIId):

[0441] [ka]

[0442] and the compound of formula (XIII) is of formula (XIIIa):

[0443] [ka]

[0444] The method of any one of sentences 21, 26, 33, 34, 60, and 61 for preparing a compound and / or a composition of any one of sentences 35, 36, 62, and 63, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene and the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex is attached to adjacent phosphates at the 3' end of the second strand.

[0445] 74. The compound of formula (XIIIa) is converted to a compound of formula (XIIIb):

[0446] [ka]

[0447] A method according to any one of sentences 70 to 73.

[0448] 75. The compound of formula (XIV) is either formula (XIVa) or formula (XIVb):

[0449] [ka]

[0450] and the compound of formula (XV) is either of formula (XVa) or formula (XIVb):

[0451] [ka]

[0452] The method of claim 69, which is dependent on sentences 70 to 73, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and (i) the RNA duplex is attached at the 5' end of the second strand to adjacent phosphates in formula (XVa), or (ii) the RNA duplex is attached at the 3' end of the second strand to adjacent phosphates in formula (XVb).

[0453] 76. Compound of formula (XII):

[0454] [ka]

[0455] [In the formula, R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl, and ethyl; R2 is hydrogen, hydroxy, -OC 1~3 Alkyl, -C(=O)OC 1~3 selected from the group consisting of alkyl, halo, and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen, and sulfur; q, r, s, t, and v are independently integers of 0 to 4, with the proviso that (i) q and r cannot both be 0 at the same time, and (ii) s, t, and v cannot all be 0 simultaneously; Z is an oligonucleoside moiety].

[0456] 77. Compound of formula (XIIa):

[0457] [ka]

[0458] 78. Compound of formula (XIIb):

[0459] [ka]

[0460] 79. Compound of formula (XIIc):

[0461] [ka]

[0462] 80. Compound of formula (XIId):

[0463] [ka]

[0464] 81. Compound of formula (XIII):

[0465] [ka]

[0466] [In the formula, R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl, and ethyl; m is an integer from 1 to 6, and n is an integer from 1 to 10.

[0467] 82. Compound of formula (XIIIa):

[0468] [ka]

[0469] 83. Compound of formula (XIIIb):

[0470] [ka]

[0471] 84. Compound of formula (XIV):

[0472] [ka]

[0473] [In the formula, R1 is selected from the group consisting of hydrogen, methyl and ethyl; R2 is hydrogen, hydroxy, -OC 1~3 Alkyl, -C(=O)OC 1~3 selected from the group consisting of alkyl, halo, and nitro; X2 is selected from the group consisting of methylene, oxygen and sulfur; s, t, and v are independently integers from 0 to 4, provided that s, t, and v cannot all be 0 at the same time.

[0474] 85. Compound of formula (XIVa):

[0475] [ka]

[0476] 86. Compound of formula (XIVb):

[0477] [ka]

[0478] 87. Compound of formula (XV):

[0479] [ka]

[0480] [In the formula, R1 at each occurrence is independently selected from the group consisting of hydrogen, methyl, and ethyl; X1 is selected from the group consisting of methylene, oxygen and sulfur; q and r are independently an integer of 0 to 4, with the proviso that q and r cannot both be 0 at the same time; Z is an oligonucleoside moiety].

[0481] 88. Compound of formula (XVa):

[0482] [ka]

[0483] 89. Compound of formula (XVb):

[0484] [ka]

[0485] 90. Use of a compound according to any one of sentences 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a compound according to any one of sentences 76, 81 to 84, and 87 for preparing a composition according to any one of sentences 30, 31, 35, 36, 57, 58, 62, and 63.

[0486] 91. Use of a compound according to sentence 85 for preparing a compound according to any one of sentences 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of sentences 30, 31, 35, 36, 57, 58, 62, and 63, wherein R2=F.

[0487] 92. Use of a compound according to sentence 86 for preparing a compound according to any one of sentences 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of sentences 30, 31, 35, 36, 57, 58, 62, and 63, wherein R2 = OH.

[0488] 93. Use of a compound according to sentence 77 for preparing a compound according to any one of sentences 20, 25, 27, 29, 54 or 56 and / or a composition according to any one of sentences 30, 31, 57 or 58.

[0489] 94. Use of a compound according to sentence 78 for preparing a compound according to any one of sentences 20, 25, 28, 29, 55 or 56 and / or a composition according to any one of sentences 30, 31, 57 or 58.

[0490] 95. Use of a compound according to sentence 79 for preparing a compound according to any one of sentences 21, 26, 32, 34, 59 or 61 and / or a composition according to any one of sentences 35, 36, 62 or 63.

[0491] 96. Use of a compound according to sentence 80 for preparing a compound according to any one of sentences 21, 26, 33, 34, 60 or 61 and / or a composition according to any one of sentences 35, 36, 62 or 63.

[0492] 97. Use of a compound according to sentence 88 for preparing a compound according to any one of sentences 20, 25, 27 to 29, 54 to 56, and / or a composition according to any one of sentences 30, 31, 57, and 58.

[0493] 98. Use of a compound according to sentence 89 for preparing a compound according to any one of sentences 21, 26, 32 to 34, 59 to 61 and / or a composition according to any one of sentences 35, 36, 62 and 63.

[0494] 99. A compound or composition obtained or obtainable by a method as set forth in any one of sentences 68 to 75.

[0495] 100. A pharmaceutical composition comprising a compound described in any one of sentences 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition described in any one of sentences 30, 31, 35, 36, 57, 58, 62, and 63, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0496] 101. A compound according to any one of sentences 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of sentences 30, 31, 35, 36, 57, 58, 62, and 63, for use in therapy.

[0497] In another aspect, the present invention may be applicable to compounds, methods, compositions, or uses in the following sections numbered 1 to 56, and any reference to any formula in a section refers only to the formula defined in sections 1 to 56. These formulas are reproduced in Figure 6. Specifically, the oligonucleoside moiety represented by Z in any of the following sections may comprise a nucleic acid for inhibiting expression of ZPI, HCII, or B4GALT1, as defined herein below.

[0498] 1. The following structure:

[0499] [ka]

[0500] [In the formula, r and s are independently an integer selected from 1 to 16; Z is an oligonucleoside moiety. A compound comprising:

[0501] 2. The compound according to claim 1, wherein s is an integer selected from 4 to 12.

[0502] 3. The compound according to paragraph 2, wherein s is 6.

[0503] 4. The compound according to any one of items 1 to 3, wherein r is an integer selected from 4 to 14.

[0504] 5. The compound according to paragraph 4, wherein r is 6.

[0505] 6. The compound according to paragraph 4, wherein r is 12.

[0506] 7. A compound according to paragraph 5, which is dependent on paragraph 3.

[0507] 8. A compound according to paragraph 6, which is dependent on paragraph 3.

[0508] 9. Z is

[0509] [ka]

[0510] [In the formula, Z1, Z2, Z3, and Z4 are independently oxygen or sulfur at each occurrence; one of the bonds between P and Z2 and between P and Z3 is a single bond, and the other is a double bond. Item 9. The compound according to any one of items 1 to 8, wherein:

[0511] 10. The compound according to any one of paragraphs 1 to 9, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0512] 11. The compound of paragraph 10, wherein the RNA compound comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, and each of the first and second strands having a 5' and a 3' end.

[0513] 12. The compound according to paragraph 11, preferably also subject to paragraphs 3 and 6, wherein the RNA compound is attached at the 5' end of its second strand to adjacent phosphates.

[0514] 13. The compound according to paragraph 11, preferably also subject to paragraphs 3 and 5, wherein the RNA compound is attached at the 3' end of its second strand to adjacent phosphates.

[0515] 14. Compounds of formula (II), preferably according to item 12:

[0516] [ka]

[0517] 15. Compounds of formula (III), preferably according to item 13:

[0518] [ka]

[0519] 16. The compound defined in any one of paragraphs 1 to 15, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2' position, preferably multiple riboses modified at the 2' position.

[0520] 17. The compound according to paragraph 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0521] 18. The compound of any one of paragraphs 1 to 17, wherein the oligonucleoside further comprises one or more degradation protecting moieties at one or more termini.

[0522] 19. The compound of paragraph 18, wherein the one or more degradation protecting moieties are not present at the terminus of an oligonucleoside chain bearing a linker / ligand moiety, and / or the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleoside is present at the distal end of the same chain relative to the end bearing a linker / ligand moiety.

[0523] 20. The compound according to any one of paragraphs 1 to 19, wherein the ligand moiety represented by formula (I) in paragraph 1 comprises one or more ligands.

[0524] 21. The compound of paragraph 20, wherein the ligand moiety of formula (I) in paragraph 1 comprises one or more carbohydrate ligands.

[0525] 22. The compound according to paragraph 21, wherein the one or more carbohydrates can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide.

[0526] 23. The compound of paragraph 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.

[0527] 24. The compound of paragraph 23, wherein the one or more carbohydrates comprise one or more N-acetyl-galactosamine moieties.

[0528] 25. The compound according to paragraph 24, comprising two or three N-acetylgalactosamine moieties.

[0529] 26. The compound of any one of the preceding clauses, wherein the one or more ligands are attached in a linear or branched configuration.

[0530] 27. The compound of paragraph 26, wherein the one or more ligands are attached in a biantennary or triantennary branched configuration.

[0531] 28. The moiety shown in formula (I) in paragraph 1:

[0532] [ka]

[0533] is any of formula (IV), (V) or (VI), preferably formula (IV):

[0534] [ka]

[0535] [In the formula, A I is hydrogen or a suitable hydroxy protecting group; a is an integer of 2 or 3, b is an integer from 2 to 5; or

[0536] [ka]

[0537] [In the formula, A I is hydrogen or a suitable hydroxy protecting group; a is an integer of 2 or 3, c and d are independently integers from 1 to 6; or

[0538] [ka]

[0539] [In the formula, A I is hydrogen or a suitable hydroxy protecting group; a is an integer of 2 or 3, e is an integer between 2 and 10. The compound according to any one of items 20 to 27, wherein

[0540] 29. The moiety shown in formula (I) in paragraph 1:

[0541] [ka]

[0542] is represented by formula (VII):

[0543] [ka]

[0544] [In the formula, A I is hydrogen, a is an integer of 2 or 3. Item 29. The compound according to any one of items 1 to 28, wherein:

[0545] 30. The compound according to paragraph 28 or 29, wherein a=2.

[0546] 31. The compound according to paragraph 28 or 29, wherein a=3.

[0547] 32. The compound according to paragraph 28, wherein b=3.

[0548] 33. Compounds of formula (VIII):

[0549] [ka]

[0550] 34. Compound of formula (IX):

[0551] [ka]

[0552] 35. The compound of paragraph 33 or 34, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2' position, preferably multiple riboses modified at the 2' position.

[0553] 36. The compound according to paragraph 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0554] 37. The compound of any one of paragraphs 33 to 36, wherein the oligonucleoside further comprises one or more degradation protecting moieties at one or more termini.

[0555] 38. The compound of paragraph 37, wherein the one or more degradation protecting moieties are not present at the terminus of an oligonucleoside chain bearing a linker / ligand moiety, and / or the one or more degradation protecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleoside is present at the distal end of the same chain relative to the end bearing a linker / ligand moiety.

[0556] 39. The compound of paragraph 33, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and wherein the RNA duplex is attached to adjacent phosphates at the 5' end of the second strand.

[0557] 40. The compound of paragraph 34, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and wherein the RNA duplex is attached to adjacent phosphates at the 3' end of the second strand.

[0558] 41. Compounds of formula (X) and (XI):

[0559] [ka]

[0560] [In the formula, r and s are independently an integer selected from 1 to 16; Z is an oligonucleoside moiety. and, if appropriate, deprotecting the ligand and / or annealing the second strand for the oligonucleoside.

[0561] 42. The compound of formula (X) is a compound of formula (Xa):

[0562] [ka]

[0563] and the compound of formula (XI) is of formula (XIa):

[0564] [ka]

[0565] 42. The method of any one of items 6, 8-14, 16-33, and 35-40, for preparing a compound of any one of items 6, 8-14, 16-33, and 35-40, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex being attached to adjacent phosphates at the 5' end of the second strand.

[0566] 43. The compound of formula (X) is a compound of formula (Xb):

[0567] [ka]

[0568] and the compound of formula (XI) is of formula (XIa):

[0569] [ka]

[0570] 42. The method of any one of items 5, 7, 9-13, 15-32, and 34-40, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having a 5' and a 3' end, and the RNA duplex being attached to adjacent phosphates at the 3' end of the second strand.

[0571] 44. The compound of formula (XIa) is converted to a compound of formula (XIb):

[0572] [ka]

[0573] 44. The method of claim 42 or 43, wherein

[0574] 45. A compound of formula (X):

[0575] [ka]

[0576] [In the formula, r is independently an integer selected from 1 to 16; Z is an oligonucleoside moiety].

[0577] 46. Compound of formula (Xa):

[0578] [ka]

[0579] 47. Compound of formula (Xb):

[0580] [ka]

[0581] 48. Compound of formula (XI):

[0582] [ka]

[0583] [In the formula, s is independently an integer selected from 1 to 16; Z is an oligonucleoside moiety].

[0584] 49. Compound of formula (XIa):

[0585] [ka]

[0586] 50. A compound of formula (XIb):

[0587] [ka]

[0588] 51. Use of a compound described in any one of paragraphs 45 and 48 to 50 for preparing a compound described in any one of paragraphs 1 to 40.

[0589] 52. Use of the compound described in item 46 for preparing a compound described in any one of items 6, 8 to 14, 16 to 33, and 35 to 40.

[0590] 53. Use of the compound described in item 47 for preparing a compound described in any one of items 5, 7, 9 to 13, 15 to 32, and 34 to 40.

[0591] 54. A compound or composition obtained or obtainable by the method according to any one of paragraphs 41 to 44.

[0592] 55. A pharmaceutical composition comprising a compound according to any one of paragraphs 1 to 40 together with a pharmaceutically acceptable carrier, diluent or excipient.

[0593] 56. A compound according to any one of paragraphs 1 to 40 for use in therapy. [Example]

[0594] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the present invention. It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended paragraphs.

[0595] Example 1: Synthesis of Tether 1 General experimental conditions: Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were visualized under UV light (254 nm) or after spraying with 5% H2SO4 in methanol (MeOH) or Stahl's ninhydrin reagent (from Sigma-Aldrich) followed by heating. Flash chromatography was performed using Biotage Sfar Silica 10, 25, 50, or 100 g columns (Uppsala, Sweden) on a Biotage Isolera One flash chromatography instrument equipped with a dual-variable UV wavelength detector (200–400 nm).

[0596] All moisture-sensitive reactions were performed 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. D-Galactosamine pentaacetate was purchased from AK scientific.

[0597] HPLC / ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer using an Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1 × 100 mm) from Waters at 60 °C. The solvent system consisted of solvent A with HO containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5 to 100% B over 15 min was used at a flow rate of 0.4 mL / min. Detector and conditions: Corona Supercharged Aerosol Detector (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0598] 1 H and 13 C NMR spectra were obtained at room temperature on a Varian spectrometer at 500 MHz ( 1 H NMR) and 125 MHz ( 13 C NMR). Chemical shifts are given in ppm and referenced to solvent residual peaks (CDCl3- 1 H NMR: δ at 7.26 ppm, and 13 C NMR δ at 77.2 ppm; DMSO-d H NMR: δ at 2.50 ppm, and 13 C NMR (δ at 39.5 ppm). Coupling constants are given in Hertz. Signal splitting patterns are reported as singlet (s), doublet (d), triplet (t) or multiplet (m).

[0599] Synthetic Route for Conjugate Building Block TriGalNAc_Tether 1:

[0600] [ka]

[0601] Preparation of Compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 equiv.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 equiv.) 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 NaHCO (100 mL) and water (100 mL). The organic layer was separated, dried over NaSO, and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0–10% MeOH in DCM over 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[0602] [ka]

[0603] Preparation of Compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 equiv.) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 equiv.) were dissolved in anhydrous DCM (40 mL) under argon, and molecular sieves 3 Å (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. TMSOTf (0.77 g, 3.49 mmol, 0.5 equiv.) was then added to the mixture, and the reaction was stirred overnight. The molecular sieves were filtered, 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 over 10 CV) to give the title product as a pale yellow oil (3.10 g, 88%, rf=0.25 (2% MeOH in DCM)). MS: C 20 H 32 N4O 11 Calculated value: 504.21. Measured value: 505.4. 1 H 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 C 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).

[0604] [ka]

[0605] 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 using a vacuum / argon cycle (3x) and hydrogenated under balloon pressure overnight. 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: C 20 H 34 N2O 11 Calculated value: 478.2. Measured value: 479.4.

[0606] [ka]

[0607] 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 DCM / water mixture (40 mL 1:1 v / v) and Na2CO3 (0.18 g, 1.7 mmol, 0.25 equiv.) was added with vigorous stirring. 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 CHCl2 (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 over 12 CV) to give the title compound as a pale yellowish oil (3.9 g, 91%, rf=0.56 (10% EtOAc in cyclohexane)). MS: C 33 H53 NO 11 Calculated value: 639.3. Measured value: 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).

[0608] [ka]

[0609] Preparation of compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 equiv.) was dissolved in CHCl (1 mL) under argon, 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 three times with toluene (5 mL) and dried under high vacuum to give the compound as its TFA salt (0.183 g, 98%). The compound was used without further purification. MS: C 21 H 29 NO 11 Calculated value: 471.6. Measured value: 472.4.

[0610] [ka]

[0611] Preparation of Compound 9: CbzNH-tris-COOH (0.72 g, 1.49 mmol, 1.0 equiv.) and GalNAc-PEG-NH (3.56 g, 7.44 mmol, 5.0 equiv.) were dissolved in N,N-dimethylformamide (DMF) (25 mL). 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 then 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 (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM over 14 CV). The product was obtained as a pale yellowish oil (1.2 g, 43%, rf=0.20 (5% MeOH in DCM)). MS: C 81 H 125 N7O 41 Calculated value: September 1852. Measured value: July 1854. 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). 13C 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).

[0612] [ka]

[0613] Preparation of Compound 10: Tri-branched GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 equiv.) was dissolved in MeOH (15 mL), and 3 drops of acetic acid (AcOH) and Pd / C (30 mg) were added. The reaction mixture was degassed using a vacuum / argon cycle (3x) and hydrogenated under balloon pressure overnight. Completion of the reaction was followed by mass analysis, and the resulting mixture was filtered through a thin pad of Celite. The solvent was evaporated, and the resulting residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellowish oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 Calculated value: 1718.8. Actual value: 1719.3.

[0614] [ka]

[0615] Preparation of Compound 11: Commercially available suberic acid bis(N-hydroxysuccinimide ester) (3.67 g, 9.9 mmol, 1.0 equiv.) was dissolved in DMF (5 mL) and triethylamine (1.2 mL) was added. To this solution was added a solution of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0 equiv.) in DMF (5 mL) dropwise. 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 over 16 CV). The product was obtained as a white solid (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM)). MS: C 15 H 23 Calculated N5O5 value: 353.4. Measured value: 354.3.

[0616] [ka]

[0617] Preparation of TriGalNAc (12): Tri-branched 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) under argon, 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 over 20 CV) to afford the title compound as a white fluffy solid (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)). MS: C 84 H 137 N 11 O 41 Calculated value: 1957.1. Actual measured value: 1959.6.

[0618] Conjugation of Tether 1 to siRNA strands: Monofluorocyclooctyne (MFCO) conjugation at the 5' or 3' end 5'-end MFCO conjugation

[0619] [ka]

[0620] 3'-end MFCO conjugation

[0621] [ka]

[0622] General conditions for MFCO conjugation: 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 to this solution was added 1 molar equivalent of a 35 mM solution of MFCO-C6-NHS ester (Berry & Associates, catalog number LK4300) in DMF. The reaction was carried out at room temperature, and after 1 hour, another 1 molar equivalent of MFCO solution was added. The reaction was allowed to proceed for another hour and monitored by LC / MS. An excess of MFCO NHS ester reagent of at least 2 molar equivalents 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 purified by reverse-phase (RP HPLC) on an Akta Pure instrument (GE Healthcare).

[0623] Purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM TEAAc pH 7, and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60°C were used. UV traces at 280 nm were recorded. A gradient of 0 to 100% B in 60 column volumes was used.

[0624] Fractions containing the full-length conjugated oligonucleotide were pooled and precipitated in a freezer with 3 M NaOAc, pH 5.2, and 85% ethanol. The collected pellet was dissolved in water. The sample was desalted by size-exclusion chromatography and concentrated using a speed-vac concentrator to give conjugated oligonucleotides in 40-80% isolated yield.

[0625] 5'-GalNAc-T1 conjugate

[0626] [ka]

[0627] 3'-GalNAc-T1 conjugate

[0628] [ka]

[0629] General procedure for TriGalNAc conjugation: MFCO-modified single chain was dissolved in water at 2000 OD / mL, and to this solution was added 1 equivalent of a solution of compound 12 (10 mM) in DMF. The reaction was carried out at room temperature, and after 3 hours, 0.7 molar equivalents of a solution of compound 12 was added. The reaction was allowed to proceed overnight, and completion was monitored by LCMS. The conjugate was diluted 15-fold with water, filtered through a 1.2 μm filter from Sartorius, and then purified by RP HPLC on an Akta Pure instrument (GE Healthcare).

[0630] RP HPLC purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM triethylammonium acetate pH 7, and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60 °C were used. UV traces at 280 nm were recorded. A gradient of 0 to 100% B in 60 column volumes was used.

[0631] Fractions containing the full-length conjugated oligonucleotide were pooled and precipitated in a freezer using 3M NaOAc, pH 5.2, and 85% ethanol. The collected pellet was dissolved in water to obtain an oligonucleotide solution of approximately 1000 OD / mL. O-acetate was removed by adding 20% aqueous ammonia. Quantitative removal of these protecting groups was confirmed by LC-MS.

[0632] The conjugates were desalted by size-exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument, giving conjugated oligonucleotides in 50–70% isolated yield.

[0633] The following scheme further illustrates the synthetic route: Scheme 1:

[0634] [ka]

[0635] Scheme 2:

[0636] [ka]

[0637] Scheme 3:

[0638] [ka]

[0639] Scheme 4:

[0640] [ka]

[0641] Scheme 5:

[0642] [ka]

[0643] Example 2: Duplex Annealing To generate the desired siRNA duplex, the two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixture was placed in a 70°C water bath for 5 minutes and then cooled to ambient temperature within 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[0644] Duplexes were analyzed by analytical SEC HPLC on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system with a Superdex™ 75 Increase 5 / 150 GL column 5 × 153–158 mm (Cytiva). The mobile phase consisted of 1× PBS containing 10% acetonitrile. An isocratic gradient was run for 10 min at a flow rate of 1.5 mL / min at room temperature. UV traces at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS, 10×, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[0645] Example 3: Synthesis of Tether 2 General experimental conditions: Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were visualized under UV light (254 nm) or after spraying with 5% H2SO4 in methanol (MeOH) or Stahl's ninhydrin reagent (from Sigma-Aldrich) followed by heating. Flash chromatography was performed using Biotage Sfar Silica 10, 25, 50, or 100 g columns (Uppsala, Sweden) on a Biotage Isolera One flash chromatography instrument equipped with a dual-variable UV wavelength detector (200–400 nm).

[0646] All moisture-sensitive reactions were performed 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. D-Galactosamine pentaacetate was purchased from AK scientific.

[0647] HPLC / ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer using an Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1 × 100 mm) from Waters at 60 °C. The solvent system consisted of solvent A with HO containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5 to 100% B over 15 min was used at a flow rate of 0.4 mL / min. Detector and conditions: Corona Supercharged Aerosol Detector (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0648] 1 H and 13 C NMR spectra were obtained at room temperature on a Varian spectrometer at 500 MHz ( 1 H NMR) and 125 MHz (13 C NMR). Chemical shifts are given in ppm and referenced to solvent residual peaks (CDCl3- 1 H NMR: δ at 7.26 ppm, and 13 C NMR δ at 77.2 ppm; DMSO-d6- 1 H NMR: δ at 2.50 ppm, and 13 C NMR (δ at 39.5 ppm). Coupling constants are given in Hertz. Signal splitting patterns are reported as singlet (s), doublet (d), triplet (t) or multiplet (m).

[0649] Synthetic Route for Conjugate Building Block TriGalNAc_Tether 2:

[0650] [ka]

[0651] Preparation of Compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 equiv.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 equiv.) 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 NaHCO (100 mL) and water (100 mL). The organic layer was separated, dried over NaSO, and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0–10% MeOH in DCM over 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[0652] [ka]

[0653] Preparation of Compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 equiv.) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 equiv.) were dissolved in anhydrous DCM (40 mL) under argon, and molecular sieves 3 Å (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. TMSOTf (0.77 g, 3.49 mmol, 0.5 equiv.) was then added to the mixture, and the reaction was stirred overnight. The molecular sieves were filtered, 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 over 10 CV) to give the title product as a pale yellow oil (3.10 g, 88%, rf=0.25 (2% MeOH in DCM)). MS: C 20 H 32 N4O 11 Calculated value: 504.21. Measured value: 505.4. 1 H 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 C 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).

[0654] [ka]

[0655] 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 using a vacuum / argon cycle (3x) and hydrogenated under balloon pressure overnight. 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: C 20 H 34 N2O 11 Calculated value: 478.2. Measured value: 479.4.

[0656] [ka]

[0657] 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 DCM / water mixture (40 mL 1:1 v / v) and Na2CO3 (0.18 g, 1.7 mmol, 0.25 equiv.) was added with vigorous stirring. 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 CHCl2 (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 over 12 CV) to give the title compound as a pale yellowish oil (3.9 g, 91%, rf=0.56 (10% EtOAc in cyclohexane)). MS: C 33 H 53 NO 11Calculated value: 639.3. Measured value: 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).

[0658] [ka]

[0659] Preparation of compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 equiv.) was dissolved in CHCl (1 mL) under argon, 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 three times with toluene (5 mL) and dried under high vacuum to give the compound as its TFA salt (0.183 g, 98%). The compound was used without further purification. MS: C 21 H 29 NO 11 Calculated value: 471.6. Measured value: 472.4.

[0660] [ka]

[0661] Preparation of Compound 9: CbzNH-tris-COOH (0.72 g, 1.49 mmol, 1.0 equiv.) and GalNAc-PEG-NH (3.56 g, 7.44 mmol, 5.0 equiv.) were dissolved in N,N-dimethylformamide (DMF) (25 mL). 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 then 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 (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM over 14 CV). The product was obtained as a pale yellowish oil (1.2 g, 43%, rf=0.20 (5% MeOH in DCM)). MS: C 81 H 125 N7O 41 Calculated value: September 1852. Measured value: July 1854. 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). 13C 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).

[0662] [ka]

[0663] Preparation of Compound 10: Tri-branched GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 equiv.) was dissolved in MeOH (15 mL), and 3 drops of acetic acid (AcOH) and Pd / C (30 mg) were added. The reaction mixture was degassed using a vacuum / argon cycle (3x) and hydrogenated under balloon pressure overnight. Completion of the reaction was followed by mass analysis, and the resulting mixture was filtered through a thin pad of Celite. The solvent was evaporated, and the resulting residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellowish oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 Calculated value: 1718.8. Actual value: 1719.3.

[0664] [ka]

[0665] Preparation of compound 14: Tri-branched GalNAc compound 10 (0.45 g, 0.26 mmol, 1.0 equiv.), HBTU (0.19 g, 0.53 mmol, 2.0 equiv.), and DIPEA (0.23 mL, 1.3 mmol, 5.0 equiv.) were dissolved in DCM (10 mL) under argon. To this mixture was added a solution of compound 13 (0.14 g, 0.53 mmol, 2.0 equiv.) in DCM (5 mL) dropwise. The reaction was stirred overnight at room temperature. The solvent was removed, and the residue was dissolved in EtOAc (50 mL), washed with water (50 mL), and dried over Na2SO4. The solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0–5% MeOH in DCM over 20 CV). The product was obtained as a white fluffy solid (0.25 g, 48%, rf=0.4 (10% MeOH in DCM)). MS: calculated for C88H137N7O42, 1965.1. Found 1965.6.

[0666] [ka]

[0667] Preparation of TriGalNAc (15): Triantennary GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 equiv.) was dissolved in EtOAc (15 mL) and Pd / C (40 mg) was added. The reaction mixture was degassed using a vacuum / argon cycle (3x) and hydrogenated under balloon pressure overnight. Completion of the reaction was monitored by mass spectrometry, and the resulting mixture was filtered through a thin pad of Celite. The solvent was removed under reduced pressure, and the resulting residue was dried under high vacuum overnight. The residue was used for conjugation to oligonucleosides without further purification (0.28 g, quantitative yield). MS:C 81 H 131 N7O 42 Calculated value: 1874.9. Actual value: 1875.3.

[0668] Conjugation of Tether 2 to the siRNA strand: TriGalNAc Tether 2 (GalNAc-T2) conjugation at the 5' or 3' end 5'-GalNAc-T2 conjugate

[0669] [ka]

[0670] 3'-GalNAc-T2 conjugate

[0671] [ka]

[0672] Preparation of TriGalNAc tether 2 NHS ester: To a solution of carboxylic acid tether 2 (compound 15, 227 mg, 121 μmol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol) were added. The solution was stirred at room temperature for 18 h and used in subsequent conjugation reactions without further purification.

[0673] General procedure for triGalNAc Tether 2 conjugation: The amine-modified single strand was dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer pH 9.6 / DMSO 4:6 (v / v), and to this solution was added 1 molar equivalent of Tether 2 NHS ester (57 mM) solution in DMF. The reaction was carried out at room temperature, and after 1 h, an additional 1 molar equivalent of NHS ester solution was added. The reaction was allowed to proceed for an additional 1 h, and reaction progress was monitored by LCMS. An excess of NHS ester reagent of at least 2 molar equivalents relative to the amino-modified oligonucleoside was required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered once through a 1.2 μm filter from Sartorius, and then purified by reverse-phase (RP HPLC) on an Akta Pure (GE Healthcare) instrument.

[0674] Purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM TEAA pH 7, and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60°C were used. The UV trace at 280 nm was recorded. A gradient of 0 to 100% B in 60 column volumes was used.

[0675] Fractions containing full-length conjugated oligonucleosides were pooled and precipitated in the freezer with 3 M NaOAc, pH 5.2, and 85% ethanol, then dissolved in water at 1000 OD / mL. O-acetate was removed with 20% ammonium hydroxide in water until completion (monitored by LC-MS).

[0676] The conjugates were desalted by size-exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument, giving conjugated oligonucleotides in 60–80% isolated yield.

[0677] The conjugate was characterized by HPLC-MS analysis using a 2.1 x 50 mm XBridge C18 column (Waters) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics). Buffer A was 16.3 mM triethylamine in 1% MeOH in HO, 100 mM HFIP, and buffer B contained 95% MeOH in buffer A. A flow rate of 250 μL / min and a temperature of 60 °C were used. UV traces at 260 and 280 nm were recorded. A gradient of 1 to 100% B within 31 min was used.

[0678] The following scheme further illustrates the synthetic route: Scheme 6:

[0679] [ka]

[0680] Scheme 7:

[0681] [ka]

[0682] Scheme 8:

[0683] [ka]

[0684] Scheme 9:

[0685] [ka]

[0686] Example 4: Duplex Annealing To generate the desired siRNA duplex, the two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixture was placed in a 70°C water bath for 5 minutes and then cooled to ambient temperature within 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[0687] Duplexes were analyzed by analytical SEC HPLC on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system with a Superdex™ 75 Increase 5 / 150 GL column 5 × 153–158 mm (Cytiva). The mobile phase consisted of 1× PBS containing 10% acetonitrile. An isocratic gradient was run for 10 min at a flow rate of 1.5 mL / min at room temperature. UV traces at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS, 10×, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[0688] Example 5: Alternative synthetic route for conjugate building block TriGalNAc_tether 2:

[0689] [ka] JPEG2025525772000122.jpg217170

[0690] Conjugation of Tether 2 to the siRNA strand: TriGalNAc Tether 2 (GalNAc-T2) conjugation at the 5' or 3' end Conjugation Conditions

[0691] [ka]

[0692] Preactivation: To a solution of compound 15 (16 μmol, 4 equiv.) in DMF (160 μL) was added TFA-O-PFP (15 μL, 21 equiv.) followed by DIPEA (23 μL, 32 equiv.) at 25° C. The tube was shaken at 25° C. for 2 h. The reaction was quenched with HO (10 μL).

[0693] Coupling: The resulting mixture was diluted with DMF (400 μl), followed by the addition of oligo-amine solution (4.0 μmol in 10× PBS, pH 7.4, 500 μL; final oligo concentration in organic and aqueous solutions: 4 μmol / ml = 4 mM). The tube was shaken at 25°C for 16 hours, and the reaction was analyzed by LCMS. The resulting mixture was treated with 28% NH4OH (4.5 ml) and shaken at 25°C for 2 hours. The mixture was analyzed by LCMS, concentrated, and purified by IP-RP HPLC to produce the oligonucleotide conjugated to Tether 2 GalNAc.

[0694] 5'-GalNAc-T2 conjugate

[0695] [ka]

[0696] 3'-GalNAc-T2 conjugate

[0697] [ka]

[0698] Example 6: Solid Phase Synthesis Method: Scale ≦1 μmol Synthesis of siRNA sense and antisense strands was performed on a MerMade 192X synthesizer using commercially available solid supports made of controlled pore glass with universal linkers (Universal CPG, with a loading of 40 μmol / g; LGC Biosearch or Glen Research).

[0699] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[0700] The 2'-O-methyl phosphoramidites used were: 5'-(4,4'-dimethoxytrityl)-N-benzoyl-adenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-cytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, isopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0701] The 2'-F phosphoramidites used were: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0702] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphoramidite, which was dissolved in DMF / MeCN (1:4, v / v). 0.02 M iodine (DNAchem) in acetonitrile / pyridine / HO was used as the oxidation reagent. Thiolation for phosphorothioate linkage was performed with 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v. 0.25 M 5-ethylthiotetrazole (ETT) in acetonitrile was used as the activation solution.

[0703] The inverted abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[0704] In each cycle, DMT was removed with a deblocking solution, 3% TCA in DCM (DNAchem).

[0705] Coupling time was 180 seconds. Oxidant contact time was set at 80 seconds, and thiolation time was 2*100 seconds.

[0706] At the end of the synthesis, the oligonucleotides were cleaved (TCI) from the solid support using a 4:1 (v / v) NH4OH:EtOH solution for 20 hours at 45° C. The solid support was then removed by filtration, the filter was washed extensively with HO, and the volume of the combined solutions was reduced by evaporation under reduced pressure.

[0707] Oligonucleotides were treated to form sodium salts by ultracentrifugation using Amicon Ultra-2 centrifugal filter units; PBS buffer (10x, Teknova, pH 7.4, sterile) or by EtOH precipitation from 1 M sodium acetate.

[0708] The identity of the single strands was assessed by MS ESI- and then annealed in water to form the final double-stranded siRNA, and the purity of the duplex was assessed by size exclusion chromatography.

[0709] Example 7: Solid Phase Synthesis Method: Scale ≥ 5 μmol Synthesis of siRNA sense and antisense strands was performed on a MerMade 12 synthesizer at a 5 μmol scale using a commercially available solid support made of controlled pore glass with a universal linker (Universal CPG, with a loading of 40 μmol / g; LGC Biosearch or Glen Research). The sense strand destined for 3' conjugation was synthesized at 12 μmol on a 3'-PT-amino-modifier C6 CPG 500 Å solid support (LGC) with a loading of 86 μmol / g.

[0710] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[0711] The 2'-O-methyl phosphoramidites used were: 5'-(4,4'-dimethoxytrityl)-N-benzoyl-adenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-cytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, isopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0712] The 2'-F phosphoramidites used were: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0713] The inverted abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[0714] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphoramidite, which was dissolved in DMF / MeCN (1:4, v / v). 0.02 M iodine (DNAchem) in acetonitrile / pyridine / HO was used as the oxidation reagent. Thiolation for phosphorothioate linkage was performed with 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v. 0.25 M 5-ethylthiotetrazole (ETT) in acetonitrile was used as the activation solution.

[0715] In each cycle, DMT was removed with a deblocking solution, 3% TCA in DCM (DNAchem).

[0716] For chains synthesized on Universal CPG, coupling was carried out with 8 equivalents of amidite for 130 seconds, with an oxidation time of 47 seconds and a thiolation time of 210 seconds.

[0717] For chains synthesized on 3'-PT-amino-modifier C6 CPG, coupling was carried out for 2*150 seconds using 8 equivalents of amidite. The oxidation time was 47 seconds and the thiolation time was 250 seconds.

[0718] At the end of the synthesis, the oligonucleotides were cleaved (TCI) from the solid support using a 4:1 (v / v) NH4OH:EtOH solution for 20 hours at 45° C. The solid support was then removed by filtration, the filter was washed extensively with HO, and the volume of the combined solutions was reduced by evaporation under reduced pressure.

[0719] The oligonucleotides were treated to form the sodium salts by EtOH precipitation from 1 M sodium acetate.

[0720] Single-stranded oligonucleotides were purified by IP-RP HPLC on an Xbridge BEH C18 5 μm, 130 Å, 19 × 150 mm (Waters) column using an increasing gradient of B in A. Mobile phase A: 240 mM HFIP, 7 mM TEA, and 5% methanol in water; mobile phase B: 240 mM HFIP, 7 mM TEA in methanol.

[0721] The purity and identity of the single strands were assessed by UPLC / MS ESI- on an Xbridge BEH C18 2.5 μm, 3 × 50 mm (Waters) column using an increasing gradient of B in A. Mobile phase A: 100 mM HFIP, 5 mM TEA in water; Mobile phase B: 20% Mobile phase A: 80% acetonitrile (v / v).

[0722] The sense strand was conjugated according to the protocol provided in either Example 1, 3 or 5.

[0723] The sense and antisense strands were then annealed in water to form the final duplex siRNA, and the purity of the duplex was assessed by size exclusion chromatography.

[0724] Example 8: Nucleic acid sequences: siRNA oligonucleosides suitable for use according to the present invention may target HCII, ZPI, and B4GALT1. The complete DNA sequences of the HCII, ZPI, and B4GALT1 targets are as follows (SEQ ID NOs: 1-3), respectively:

[0725] SEQ ID NO: 1 (HCII)

[0726] JPEG2025525772000126.jpg92168JPEG2025525772000127.jpg255166JPEG2025525772000128.jpg255168 JPEG2025525772000129.jpg255167JPEG2025525772000130.jpg255166JPEG2025525772000131.jpg118167

[0727] Sequence number 2 (ZPI)

[0728] JPEG2025525772000132.jpg118167JPEG2025525772000133.jpg255168JPEG2025525772000134.jpg25516 7JPEG2025525772000135.jpg255168JPEG2025525772000136.jpg255167JPEG2025525772000137.jpg41165

[0729] SEQ ID NO: 3 (B4GALT1)

[0730] JPEG2025525772000138.jpg196168JPEG2025525772000139.jpg255166JPEG2025525772000140.jpg255167JPEG2025525772000141.jpg25516 7JPEG2025525772000142.jpg255167JPEG2025525772000143.jpg255168JPEG2025525772000144.jpg255167JPEG2025525772000145.jpg25516 6JPEG2025525772000146.jpg255168JPEG2025525772000147.jpg255166JPEG2025525772000148.jpg255167JPEG2025525772000149.jpg2551 68JPEG2025525772000150.jpg255167JPEG2025525772000151.jpg255167JPEG2025525772000152.jpg252166JPEG2025525772000153.jpg9165

[0731] Table 1 below provides the oligonucleoside mRNA target sequences for HCII, ZPI, and B4GALT1, along with the corresponding positions in the transcripts NM_000185.4 (HCII), NM_016186.3 (ZPI), and NM_001497.4 (B4GALT1).

[0732] [Table 1]

[0733] Table 2 provides the unmodified first (antisense) strand sequences and corresponding unmodified second (sense) strand sequences for siRNA oligonucleosides according to the present invention (targeting HCII, ZPI and B4GALT1) along with their corresponding positions in the entire gene sequence of SEQ ID NO: 1, 2 or 3, as follows:

[0734] [Table 2]

[0735] Table 3 provides modified first (antisense) sequences for siRNA oligonucleosides according to the present invention (targeting HCII, ZPI and B4GALT1) along with the corresponding unmodified first (antisense) sequences, as follows:

[0736] [Table 3] JPEG2025525772000157.jpg253168JPEG2025525772000158.jpg201167

[0737] Table 4 provides modified second (sense) sequences for siRNA oligonucleosides according to the present invention (targeting HCII, ZPI and B4GALT1) along with the corresponding unmodified second (sense) sequences, as follows:

[0738] [Table 4]

[0739] Some of the modified second strand sequences exemplified above in Table 4 include the preferred 5'iaia motif, however, it should be understood that the scope of these modified second strand sequences also includes Me / F modified second strands in the absence of the 5'iaia motif.

[0740] Table 5 identifies duplexes using duplex IDs with reference to modified antisense and sense IDs from Tables 3 and 4 above.

[0741] [Table 5] JPEG2025525772000161.jpg36167

[0742] Definitions provided in the table above: A - Adenosine C - cytidine G - Guanosine T - thymidine m - 2'-O-methyl f - 2'fluoro s - phosphorothioate linkage ia - inverted abasic nucleoside

[0743] Example 9: Inhibition screening for target gene expression in human Huh7 cells Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB Cell Bank) are maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO atmosphere. Cells are transfected with siRNA duplexes targeting target gene mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 115), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 114)) at final duplex concentrations of 5 nM and 0.1 nM. Transfection is performed by adding 9.7 μL of Opti-MEM (ThermoFisher) + 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture is incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells are incubated at 37°C / 5% CO2 for 24 hours, after which total RNA is purified using an RNeasy 96 Kit (Qiagen). Each duplex is tested by transfection in duplicate wells in two independent experiments.

[0744] cDNA synthesis was performed using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) was performed using the FastStart Universal Probe Master Kit (Roche) with primers specific for the target gene and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or ABI QuantStudio 7.

[0745] qPCR is performed in duplicate on the cDNA from each well, and the average Ct is calculated. Relative HCII expression is calculated from the average Ct value, normalized to GAPDH, relative to untreated cells using the comparative Ct (ΔΔCt) method. Based on the results of the primary screening, siRNA duplexes that show good activity are selected for dose-response follow-up.

[0746] Example 10: Dose response for target gene expression in human Huh7 cells Huh7 cells (a human hepatocyte-derived cell line obtained from the JCRB Cell Bank) are maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO atmosphere. Cells are transfected with siRNA duplexes targeting target gene mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 115), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 114)) using 10x3-fold serial dilutions spanning a final duplex concentration range of 20 nM to 1 pM. Transfections are performed by adding 9.7 μL of Opti-MEM (ThermoFisher) plus 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture is incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells are incubated at 37°C / 5% CO2 for 24 hours, after which total RNA purification is performed using an RNeasy 96 Kit (Qiagen). Each duplex is tested by transfection in duplicate wells in a single experiment.

[0747] cDNA synthesis was performed using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) was performed using the FastStart Universal Probe Master Kit (Roche) with primers specific for the target gene and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or ABI QuantStudio 7.

[0748] qPCR is performed in duplicate on cDNA from each well, and the average Ct is calculated. Relative HCII expression is calculated from the average Ct values using the comparative Ct (ΔΔCt) method, normalized to GAPDH, and relative to untreated cells. The maximum percent inhibition of HCII expression and IC50 values are calculated using a four-parameter (variable slope) model using GraphPad Prism 9.

[0749] Example 10: Dose response for inhibition of ZPI and B4GALT1 in human Huh7 cells Huh7 cells (a human hepatocyte-derived cell line obtained from the JCRB Cell Bank) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO2 atmosphere. Cells were transfected with 0.1 nM and 1 nM siRNA duplexes designed against the target or negative control siRNA. Transfection was performed by adding 9.7 μL of Opti-MEM (ThermoFisher) plus 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells were incubated at 37°C / 5% CO2 for 24 hours, after which total RNA purification was performed using the RNeasy 96 Kit (Qiagen). Each duplex was tested by transfection in duplicate wells, and the experiment was repeated three times.

[0750] cDNA synthesis was performed using the FastKing RT kit (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) was performed using the TaqMan Gene Expression Assay Kit (ThermoFisher Scientific) with primers specific for human B4GALT1 (Hs00155245_m1), human ZPI (Hs01547819_m1), and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or ABI QuantStudio 7.

[0751] qPCR was performed in duplicate on cDNA from each well, and the average Ct was calculated. Relative target expression was calculated from the average Ct values, normalized to GAPDH, relative to untreated cells using the comparative Ct (ΔΔCt) method.

[0752] siRNA duplexes ETXM1200, ETXM1203, ETXM1204, ETXM1205, ETXM1206, and ETXM1207 were tested for inhibition of ZPI (Figure 9). siRNA duplexes ETXM1217, ETXM1766, ETXM1767, ETXM1768, ETXM1769, and ETXM1770 (Figure 10) and ETXM1218, ETXM1774, ETXM1775, ETXM1776, ETXM1777, and ETXM1778 (Figure 11) were tested for inhibition of B4GALT1.

[0753] It is not intended that the present invention be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the descriptions and teachings herein. Such variations can be made without departing from the true scope and spirit of the disclosure and are intended to be within the scope of the disclosure.

[0754] In the event that an ambiguity exists between a sequence herein and a sequence in the accompanying sequence listing, the sequence provided herein shall be considered to be the correct sequence.

Claims

1. A nucleic acid for inhibiting the expression of a target gene, 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 first and second strands form a double-stranded region with a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): him-him-(Me) 8 -(F) 3 -(Along with) 10 [In the formula, ia represents an inverted baseless nucleoside.] A nucleic acid comprising a 2' sugar and base-free modification pattern, wherein the nucleoside of the first chain comprises a 2' sugar modification pattern, wherein the modification is selected from at least 2'Me and 2'F sugar modifications, provided that the total number of 2'F sugar modifications in the first chain is not composed of 4 or 6 2'F modifications.

2. The nucleic acid according to claim 1, wherein the nucleoside of the first chain includes a 2' sugar modification pattern, the modification is selected from at least 2'Me and 2'F sugar modifications, and the total number of 2'F sugar modifications in the first chain consists of 3, 5, or 7 2'F modifications.

3. The nucleoside of the first chain contains a 2' sugar modification pattern, the modification is selected from at least 2'Me and 2'F sugar modifications, and the total number of 2'F sugar modifications in the first chain consists of 3 2'F modifications. The nucleosides of the first chain are as follows (5'-3'): Me-F-(Me) 3 -X 1 -(Me) 7 -F-Me-F-(Me) 7 [In the formula, X1 is a thermal destabilization modification.] The nucleic acid according to claim 2, comprising the 2' sugar modification pattern.

4. The nucleoside of the first chain contains a 2' sugar modification pattern, the modification is selected from at least 2'Me and 2'F sugar modifications, and the total number of 2'F sugar modifications in the first chain consists of 5 2'F modifications. (a) The nucleoside of the first chain is (5'-3'): Me-F-Me-X 2 -Me-F-(Me) 7 -(F-Me) 2 -X 3 -Me-X 4 -(Me) 3 [In the formula, X2, X3, and X4 are selected from 2'Me and 2'F glycosylation, where at least one of X2, X3, and X4 is a 2'F glycosylation and the other two are 2'Me glycosylation.] It includes the 2' sugar modification pattern, or (b) The nucleoside of the first chain is (5'-3'): Me-F-(Me) 3 -X 1 -Me-(F) 2 -(Me) 4 -F-Me-F-(Me) 7 [In the formula, X1 is a thermal destabilization modification.] The nucleic acid according to claim 2, comprising the 2' sugar modification pattern.

5. The nucleoside of the first chain contains a 2' sugar modification pattern, the modification is selected from at least 2'Me and 2'F sugar modifications, and the total number of 2'F sugar modifications in the first chain consists of 7 2'F modifications. The nucleosides of the first chain are as follows (5'-3'): Me-F-Me-X 2 -Me-F-Me-(F) 2 -(Me) 4 -(F-Me) 2 -X 3 -Me-X 4 -(Me) 3 [In the formula, X2, X3, and X4 are selected from 2'Me and 2'F glycosylation, where at least one of X2, X3, and X4 is a 2'F glycosylation and the other two are 2'Me glycosylation.] The nucleic acid according to claim 2, comprising the 2' sugar modification pattern.

6. (i)X 2 This is a 2'F sugar modification, and X 3 and X 4 This is a 2'Me sugar modification, (ii) X 3 is 2'F glycosylated, and X 2 and X 4 are 2'Me glycosylated, or (iii) The nucleic acid according to claim 4, wherein X4 is 2'F glycosylated and X2 and X3 are 2'Me glycosylated.

7. (i) X 2 is a 2'F sugar modification, X 3 and X 4 is a 2'Me sugar modification, (ii) X 3 is 2'F glycosylated, and X 2 and X 4 are 2'Me glycosylated, or (iii) The nucleic acid according to claim 5, wherein X4 is 2'F glycosylated and X2 and X3 are 2'Me glycosylated.

8. The nucleic acid according to claim 1, wherein two phosphorothioate nucleoside links are located between three consecutive positions in the 5'-terminal region of the second strand, the first phosphorothioate nucleoside link is located between the first base nucleoside and the adjacent second base nucleoside in the 5'-terminal region of the second strand when read from the 5' end, and the second phosphorothioate nucleoside link is located between the second base nucleoside and the adjacent third base nucleoside in the 5'-terminal region of the second strand.

9. The nucleic acid according to claim 1, wherein two phosphorothioate nucleoside linkages are located between three consecutive positions in both the 5' and 3' terminal regions of the first strand, so that the terminal nucleosides in the 5' and 3' terminal regions of the first strand are attached by phosphorothioate nucleoside linkages to the second-to-last adjacent nucleosides in the respective 5' and 3' regions, and the second-to-last nucleosides in the respective 5' and 3' regions are attached by phosphorothioate nucleoside linkages to the third-to-last adjacent nucleosides in the respective 5' and 3' regions.

10. A nucleic acid for inhibiting the expression of a target gene, comprising a first strand at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region having a length of at least 17 nucleosides. (a) The nucleoside of the second chain is (5'-3'): him-him-(Me) 8 -(F) 3 -(Along with) 10 [In the formula, ia represents an inverted baseless nucleoside.] The 2' sugar and base-free modification pattern is included, and the nucleoside of the first chain is (5'-3'): Me-F-(Me) 3 -X 1 -(Me) 7 -F-Me-F-(Me) 7 [In the formula, X 1 This is a thermal destabilization modification. (Me-F) 3 -(Me) 7 -F-Me-F-(Me) 7 Me-F-(Me) 3 -F-(Me) 7 -(F-Me) 2 -F-(Me) 5 Me-F-(Me) 3 -F-(Me) 7 -F-Me-F-(Me) 3 -F-(Me) 3 Me-F-(Me) 3 -X 1 -Me-(F) 2 -(Me) 4 -F-Me-F-(Me) 7 [In the formula, X 1 This is a thermal destabilization modification. (Me-F) 3 -Me-(F) 2 -(Me) 4 -(F-Me) 2 -(Me) 6 Me-F-(Me) 3 -F-Me-(F) 2 -(Me) 4 -(F-Me) 2 -F-(Me) 5 Me-F-(Me) 3 -F-Me-(F) 2 -(Me) 4 -(F-Me) 2 -(Me) 2 -F-(Me) 3 It includes a 2' sugar modification pattern selected from one of the following, or (b) The nucleoside of the second chain is (5'-3'): ia-ia-Me(s)Me(s)(Me) 6 -(F) 3 -(Me) 10 [In the formula, ia represents an inverted baseless nucleoside, and (s) represents a phosphorothioate linkage.] The 2' sugar and base-free modification pattern is included, and the nucleoside of the first chain is (5'-3'): Me(s)F(s)(Me) 3 -X 1 -(Me) 7 -F-Me-F-(Me) 5 (s)Me(s)Me [wherein X 1 is a thermal destabilization modification] Me(s)F(s)Me-F-Me-F-(Me) 7 -F-Me-F-(Me) 5 (s)Me(s)Me Me(s)F(s)(Me) 3 -F-(Me) 7 -(F-Me) 2 -F-(Me) 3 (s)Me(s)Me Me(s)F(s)(Me) 3 -F-(Me) 7 -F-Me-F-(Me) 3 -F-Me(s)Me(s)Me Me(s)F(s)(Me) 3 -X 1 -Me-(F) 2 -(Me) 4 -F-Me-F-(Me) 5 (s)Me(s)Me [wherein X 1 is a thermal destabilization modification] Me(s)F(s)Me-F-Me-F-Me-(F) 2 -(Me) 4 -(F-Me) 2 -(Me) 4 (s)Me(s)Me Me(s)F(s)(Me) 3 -F-Me-(F) 2 -(Me) 4 -(F-Me) 2 -F-(Me) 3 (s)Me(s)Me Me(s)F(s)(Me) 3 -F-Me-(F) 2 -(Me) 4 -(F-Me) 2 -(Me) 2 -F-Me(s)Me(s)Me A nucleic acid containing a 2' sugar modification pattern selected from one of the following.

11. The nucleic acid according to claim 1, wherein two consecutive unbase nucleosides in the 5' terminal region of the second strand include an unbase nucleoside that is the terminal nucleoside in the 5' terminal region of the second strand, and the other unbase nucleoside is the second to last nucleoside in the 5' terminal region of the second strand, and (a) the second to last unbase nucleoside is connected to the adjacent first base nucleoside in the adjacent 5' terminal region via a reverse nucleoside linkage, (b) the reverse linkage is a 5-5' reverse linkage, and (c) the linkage between the terminal unbase nucleoside and the second to last unbase nucleoside is 3'-5' when read toward the end containing the terminal unbase nucleoside and the second to last unbase nucleoside.

12. The nucleic acid according to claim 1, wherein the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties in the 3' terminal region of the second strand.

13. The ligand part is One or more N-acetylgalactosamine (GalNAc) ligands, and / or One or more N-acetylgalactosamine (GalNAc) ligand derivatives, and / or One or more N-acetylgalactosamine (GalNAc) ligands and / or derivatives thereof conjugated to nucleic acids via a linker. The nucleic acid according to claim 12, comprising:

14. (a) The nucleic acid has structure: 【Chemistry 1】 [In the formula, R 1 Each time it appears, it is independently selected from the group consisting of hydrogen, methyl, and ethyl. R 2 is hydrogen, hydroxyl, -OC 1～3 Alkyl, -C(=O)OC 1～3 Selected from the group consisting of alkyl, halo, and nitro, X 1 and X 2 Each time it appears, it is independently selected from the group consisting of methylene, oxygen, and sulfur. m is an integer between 1 and 6. n is an integer between 1 and 10. q, r, s, t, and v are independent integers between 0 and 4, however, (i) q and r cannot both be 0 at the same time, and (ii) s, t, and v cannot all be 0 at the same time. Z is an oligonucleoside. Having, or (b) Nucleic acid structure: 【Chemistry 2】 [In the formula, r and s are independent integers selected from 1 to 16. Z is an oligonucleoside. The nucleic acid according to claim 13, having the following characteristics.

15. The nucleic acid according to claim 1, wherein the nucleic acid is an siRNA oligonucleoside.

16. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 15 in combination with a pharmaceutically acceptable excipient or carrier.

17. A pharmaceutical composition according to claim 16 for use in treatment.

18. The pharmaceutical composition according to claim 16, for use in the prevention or treatment of diseases related to impaired hemostasis, such as hemophilia.

19. A pharmaceutical composition according to claim 16 for use in the prevention or treatment of diabetes.

20. The pharmaceutical composition according to claim 16, for use in the prevention or treatment of cardiovascular disease.

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