Nucleic acid compound

Abstract

The present invention provides novel nucleic acid compounds suitable for therapeutic use. Further, the present invention provides methods for making these compounds, as well as 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. Further, the present invention provides methods for making these compounds and methods for using the compounds for treating various diseases and conditions.

Background Art

[0002] Nucleic acid compounds have important therapeutic applications in medicine. Nucleic acids can be used to silence genes that are the cause of a particular disease. Gene silencing prevents the formation of proteins by inhibiting translation. Importantly, gene silencing agents are promising alternatives to conventional small organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleotide / oligoside that prevent protein formation by gene silencing.

[0003] Several modified siRNA compounds, including siRNA / RNAi therapeutic agents for treating various diseases including central nervous system diseases, inflammatory diseases, metabolic disorders, oncology, infectious diseases, and eye diseases, have been developed particularly in the past 20 years for diagnostic and therapeutic purposes.

Summary of the Invention

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

[0005] Description of the Invention A nucleic acid for inhibiting the 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 double-stranded region having a length of at least 17 nucleosides, The second strand contains two consecutive abasic nucleosides in the 5'-terminal region of the second strand, which includes the 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, (a) the penultimate abasic nucleoside is connected to the adjacent first base nucleoside in the adjacent 5'-terminal proximal region through a reverse nucleoside internucleoside linkage, (b) the reverse linkage is a 5-5' reverse linkage, and (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when read towards the end including the terminal abasic nucleoside and the penultimate abasic nucleoside. A nucleic acid, wherein the first strand contains at least one duplex thermal destabilizing modification within the first 9 nucleoside positions of its 5'-region.

[0006] A nucleic acid for inhibiting the 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 having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 [wherein, ia represents a reversed abasic nucleoside] and includes a 2'-sugar and abasic modification pattern of, a nucleic acid, wherein the first strand contains at least one duplex thermal destabilizing modification within the first 9 nucleoside positions of its 5'-region.

[0007] The nucleic acid described herein, wherein the destabilizing modification is selected from unlocked nucleic acid (UNA) and glycol nucleic acid (GNA).

[0008] The nucleic acid described herein, wherein the destabilizing modification includes at least one unlocked nucleic acid (UNA).

[0009] A nucleic acid described herein, wherein the destabilizing modification comprises at least one glycol nucleic acid (GNA).

[0010] The nucleic acid described herein, wherein the at least one double-stranded thermal destabilizing modification is at position 2 to 9, preferably position 2 to 8, more preferably position 3 to 8, more preferably position 4 to 8, most preferably position 6 or 7 of the nucleoside in the 5' region of the first strand.

[0011] The nucleic acid described herein, wherein the nucleoside of the first strand comprises a 2'-sugar modification pattern, 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 strand is not composed of 4 or 6 2'-F modifications.

[0012] The nucleic acid described herein, wherein the nucleoside of the first strand comprises 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 strand consists of 3, 5 or 7 2'-F modifications.

[0013] The nucleic acid described herein, wherein the nucleoside of the first strand comprises 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 strand consists of 3 2'-F modifications.

[0014] The nucleoside of the first strand is as follows (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 [wherein X1 is a thermal destabilizing modification] The nucleic acid described herein, which comprises the 2'-sugar modification pattern of.

[0015] The nucleic acid described herein, wherein the nucleoside of the first strand comprises 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 strand consists of 5 2'-F modifications.

[0016] The nucleosides of the first strand are as follows (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 [wherein X1 is a heat destabilizing modification] A nucleic acid as described herein, comprising the 2'-sugar modification pattern of

[0017] The nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consisting of 7 2'-F modifications, a nucleic acid as described herein.

[0018] Two phosphorothioate internucleoside linkages are each present between three consecutive positions in the region near the 5'-end of the second strand, the first phosphorothioate internucleoside linkage being present between the first base nucleoside and the adjacent second base nucleoside, when read from the 5'-end, in the region near the 5'-end of the second strand, and the second phosphorothioate internucleoside linkage being present between the second base nucleoside and the adjacent third base nucleoside in the region near the 5'-end of the second strand, a nucleic acid as described herein.

[0019] Two phosphorothioate internucleoside linkages are each present between three consecutive positions in both the 5'-end region and the 3'-end region of the first strand, whereby the terminal nucleosides in each of the 5'-end region and the 3'-end region of the first strand are each attached by a phosphorothioate internucleoside linkage to the respective 5'- and 3'-adjacent second last nucleosides, and the respective 5'- and 3'-second last nucleosides are attached by a phosphorothioate internucleoside linkage to the respective 5'- and 3'-adjacent third last nucleosides, a nucleic acid as described herein.

[0020] A nucleic acid for inhibiting the 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 double-stranded region having a length of at least 17 nucleosides, 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] including a 2'-sugar and abasic modification pattern of, and the nucleosides of the first strand are as follows (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 [wherein, X1 is a heat destabilizing modification], or Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 [wherein, X1 is a heat destabilizing modification] A nucleic acid including a 2'-sugar modification pattern selected from one of the above.

[0021] A nucleic acid for inhibiting the 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 double-stranded region having a length of at least 17 nucleosides, 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] including a 2'-sugar and abasic modification pattern of, and the nucleosides of the first strand are as follows (5'-3'): Me(s)F(s)(Me)3-X1-(Me)7-F-Me-F-(Me)5(s)Me(s)Me [wherein, X1 is a heat destabilizing modification], or Me(s)F(s)(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)5(s)Me(s)Me [wherein, X1 is a heat destabilizing modification] A nucleic acid comprising a 2'-sugar modification pattern selected from one of them.

[0022] The first strand comprises at least 17 consecutive nucleosides that differ from any one of the sequences of the first strand listed in Table 2 by 0 or 1 nucleoside. In particular, the first strand comprises nucleosides 2-18 of any one of the sequences defined in Table 2. The nucleic acid described herein.

[0023] The nucleic acid according to the present invention may further comprise a first strand comprising at least 17 consecutive nucleosides that differ from any one of the sequences of the first strand listed in Table 3 by 0 or 1 nucleoside. In particular, the first strand comprises nucleosides 2-18 of any one of the sequences defined in Table 3.

[0024] The second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of the sequences of the second strand listed in Table 2 by 0 or 1 nucleoside, and the double-stranded region comprises at least 14, 15, 16 or 17 complementary base pairs. The nucleic acid described herein.

[0025] The nucleic acid according to the present invention may further comprise a second strand comprising a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of the sequences of the second strand listed in Table 4 by 0 or 1 nucleoside, and the double-stranded region comprises at least 14, 15, 16 or 17 complementary base pairs.

[0026] The first strand comprises any one of the sequences of the first strand listed in Table 2, and / or the second strand comprises any one of the sequences of the second strand listed in Table 2. The nucleic acid described herein.

[0027] The first strand comprises any one of the sequences of the first strand listed in Table 3, and / or the second strand comprises any one of the sequences of the second strand listed in Table 4. The nucleic acid described herein.

[0028] A nucleic acid as described herein, wherein the first strand and the second strand form any one of the double strands listed in Table 5.

[0029] A nucleic acid as described herein, wherein the nucleic acid is an siRNA oligonucleoside.

[0030] 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 base nucleoside in the adjacent 5'-terminal proximal region through a reverse nucleoside internucleoside linkage, (b) the reverse linkage is a 5-5' reverse linkage, and (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when read towards the end including the terminal abasic nucleoside and the penultimate abasic nucleoside.

[0031] A nucleic acid according to the invention, wherein the nucleic acid is directly or indirectly conjugated to one or more ligand moieties, and optionally, the ligand moiety is present in the terminal region of the second strand, typically its 3'-terminal region, and typically, 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 their derivatives conjugated to the nucleic acid through a linker may be included. Typically, one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly conjugated to the 5' or 3'-terminal region of the second strand of the nucleic acid, typically in its 3'-terminal region.

[0032] The following structure:

[0033]

Chemical formula

[0034] A nucleic acid according to the invention, comprising a ligand moiety comprising

[0035] The following structure:

[0036]

Chemical formula

[0037] [wherein, each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl and ethyl, R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo and nitro, each occurrence of X1 and X2 is 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, v are independently integers from 0 to 4, provided that both q and r cannot 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 invention, comprising a ligand moiety comprising

[0038] Structure

[0039]

Chemical formula

[0040] [wherein, [oligonucleotide] represents consecutive nucleosides of the second strand] A nucleic acid according to the invention, comprising

[0041] Or, the following structure:

[0042] [Chemical formula]

[0043] [wherein, r and s are independently integers selected from 1 to 16, and Z is an oligonucleoside] A nucleic acid according to the invention, comprising a ligand moiety comprising

[0044] Structure

[0045] [Chemical formula]

[0046] [wherein, [oligonucleotide] represents consecutive nucleosides of the second strand] A nucleic acid according to the invention, comprising

[0047] The present invention further provides a pharmaceutical composition comprising the nucleic acid described herein in combination with a pharmaceutically acceptable excipient or carrier.

[0048] The present invention further provides the nucleic acid or pharmaceutical composition described herein for use in therapy.

[0049] The present invention further provides the nucleic acid or pharmaceutical composition described herein for use in the prevention or treatment of a disease related to a hemostatic disorder, such as a disease related to a hemostatic disorder such as hemophilia.

[0050] The present invention further provides the nucleic acid or pharmaceutical composition described herein for use in the prevention or treatment of diabetes.

[0051] The present invention further provides the nucleic acid or pharmaceutical composition described herein for use in the prevention or treatment of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0052]

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Figure 10

Mode for Carrying Out the Invention

[0053] Definition As used herein, the "first strand," also referred to herein as the antisense strand or guide strand and used interchangeably herein, refers to a nucleic acid strand, such as an siRNA strand, such as a strand of dsiRNA, that includes a region that is substantially complementary to a target sequence, such as mRNA. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence. When the complementary region is not completely complementary to the target sequence, mismatches can typically be in the internal or terminal regions of the molecule. In some embodiments, the double-stranded nucleic acids of the invention, such as siRNA agents, include nucleoside mismatches in the antisense strand.

[0054] The "second strand" (also referred to herein as the sense strand or passenger strand and used interchangeably herein) refers to a strand of a nucleic acid, such as an siRNA, that includes a region that is substantially complementary to a region of the antisense strand as defined herein.

[0055] In the context of a molecule comprising a nucleic acid optionally further comprising a linker moiety and having a ligand moiety, the nucleic acids of the invention may be referred to as oligonucleosides or oligonucleoside moieties.

[0056] An oligonucleotide is a short nucleic acid polymer. Oligonucleotides contain phosphodiester bonds between their nucleoside components (base + sugar), but the present invention is not limited to oligonucleotides that are always linked by such phosphodiester bonds between adjacent nucleosides, and other oligomers of nucleosides linked by bonds that are other than phosphodiester bonds are contemplated. For example, the bond between nucleosides can be a phosphorothioate bond. Thus, the term "oligonucleoside," as used herein, encompasses both oligonucleotides and other oligomers of nucleosides. Oligonucleosides that are nucleic acids having at least a portion that is an oligonucleotide are preferred according to the present invention. Oligonucleosides having one or more, or 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 bonds between nucleosides and also having one or more phosphorothioate backbone bonds between nucleosides (typically in the terminal regions of the first and / or second strands) are also preferred according to the present invention.

[0057] 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. Thus, in all cases where the present application refers to an oligonucleotide, particularly in the chemical structures disclosed herein, the oligonucleotide can similarly be an oligonucleoside as defined herein.

[0058] In some embodiments, the double-stranded nucleic acid of the present invention, such as an siRNA agent, comprises a nucleoside mismatch in the sense strand. In some embodiments, the nucleoside mismatch is, for example, within 5, 4, 3, 2, or 1 nucleoside from the 3' end of the nucleic acid, such as an siRNA.

[0059] In another embodiment, the nucleoside mismatch is, for example, at the 3' terminal nucleoside of the nucleic acid, such as an siRNA.

[0060] The "target sequence" (which may also be referred to as target RNA or target mRNA) refers to a continuous portion of the nucleoside sequence of an mRNA molecule formed during gene transcription, including the mRNA that is the product of RNA processing of the primary transcript.

[0061] 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 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleosides in length. Ranges and lengths intermediate to those listed above are also contemplated as part of the present invention.

[0062] The term "ribonucleoside" or "nucleoside" may also refer to modified nucleosides, which are further detailed below.

[0063] The nucleic acid may be DNA or RNA and may contain modified nucleosides. RNA is the preferred nucleic acid.

[0064] The terms "iRNA", "siRNA", "RNAi agent", and "iRNA agent", "RNA interference agent" when used interchangeably herein, refer to agents that contain RNA and mediate the 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).

[0065] Double-stranded RNA is referred to herein as "double-stranded siRNA (dsiRNA) agent", "double-stranded siRNA (dsiRNA) molecule", "double-stranded RNA (dsRNA) agent", "double-stranded RNA (dsRNA) molecule", "dsiRNA agent", "dsiRNA molecule", or "dsiRNA", which refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two anti-parallel and substantially complementary nucleic acid strands that are said to have "sense" and "antisense" orientations with respect to the target RNA.

[0066] The majority of the nucleosides of each strand of a nucleic acid, such as a dsiRNA molecule, are preferably ribonucleosides, but in that case, each or both strands can also contain one or more non-ribonucleosides, such as deoxyribonucleosides or modified nucleosides. Further, as used herein, "siRNA" can include ribonucleosides having chemical modifications.

[0067] The term "modified nucleoside" independently refers to a nucleoside having a modified sugar moiety, a modified internucleoside linkage, or a modified nucleic acid base, or any combination thereof. Thus, the term modified nucleoside encompasses substitutions, additions, or removals to the internucleoside linkage, sugar moiety, or nucleic acid base, for example, functional groups or atoms. Any such modification, when used in an siRNA-type molecule, is encompassed by "iRNA" or "RNAi agent" or "siRNA" or "siRNA agent" for the purposes of this specification and the claims.

[0068] The two strands forming the double-stranded structure may be different portions of one larger molecule, or they may be separate molecules, such as RNA molecules.

[0069] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the double-stranded structure of the nucleic acid according to the present invention. The nucleic acid according to the present invention may include an overhang of at least one nucleoside; alternatively, the overhang may include at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more. The nucleoside overhang may include or consist of nucleoside / nucleoside analogs, including deoxynucleosides. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleoside(s) of the overhang may be present at the 5'-end, 3'-end, or both ends of either the antisense or sense strand.

[0070] In certain embodiments, the antisense strand has an overhang of 1 to 10 nucleosides, such as 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 at the 3'- or 5'-end.

[0071] "Blunt" or "blunt end" means that there are no unpaired nucleosides at the ends of the double-stranded nucleic acid, i.e., no nucleoside overhang. The nucleic acids of the present invention include those having no nucleoside overhang at one end or no nucleoside overhang at either end.

[0072] 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 containing the first nucleoside sequence to hybridize under certain conditions with an oligonucleoside containing the second nucleoside sequence to form a double-stranded structure, as would be understood by one of ordinary 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, 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").

[0073] Complementary sequences within a nucleic acid, such as a dsiRNA, as described herein, include base pairing of an oligonucleoside containing a first nucleoside sequence with an oligonucleoside containing a second nucleoside sequence over the entire length of one or both of the nucleoside sequences. Such sequences can be said to be "fully complementary" to each other herein. However, when a first sequence is said to be "substantially complementary" or "partially complementary" to a second sequence herein, 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 the conditions most relevant to their ultimate application, such as inhibition of gene expression via the RISC pathway. Overhangs shall not be considered mismatches with respect to determination of complementarity. For example, a nucleic acid, such as a dsiRNA, containing one oligonucleoside 17 nucleosides in length and another oligonucleoside 19 nucleosides in length, wherein the longer oligonucleoside contains a 17-nucleoside sequence that is fully complementary to the shorter oligonucleoside, can still be said to be "fully complementary."

[0074] A "complementary" array, as used herein, may also include, or be entirely formed from, non-Watson-Crick base pairs, or base pairs formed from non-natural and modified nucleosides, so long as the above requirements regarding its ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0075] The terms "complementary", "fully complementary" and "substantially / partially complementary" as used herein can be used in relation to base matching between nucleic acids, for example between the sense and antisense strands of dsiRNA, or between the antisense strand of a double-stranded nucleic acid, such as an siRNA agent, and a target sequence.

[0076] In the present invention, the second strand of the 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 the nucleic acid according to the present invention are partially complementary when they form a double-stranded region having a length of at least 17 base pairs and containing no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0077] In certain embodiments, the first and second strands of the nucleic acid according to the present invention are partially complementary when they form a double-stranded region having a length of 19 base pairs and containing no more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, the first and second strands of the nucleic acid according to the present invention are partially complementary when they form a double-stranded region having a length of 21 base pairs and containing no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0078] Alternatively, the first and second strands of the nucleic acid according to the present invention are partially complementary when they form a double-stranded region having a length of at least 17 base pairs, and at least 14, 15, 16 or 17 of said base pairs are complementary base pairs, particularly Watson-Crick base pairs.

[0079] In certain embodiments, the first and second strands of the nucleic acids according to the invention form a double-stranded region having a length of 19 base pairs, and are partially complementary when at least 14, 15, 16, 17, 18 or all 19 base pairs are complementary base pairs, in particular Watson-Crick base pairs. In certain embodiments, the first and second strands of the nucleic acids according to the invention form a double-stranded region having a length of 21 base pairs, and are partially complementary when at least 16, 17, 18, 19, 20 or all 21 base pairs are complementary base pairs, in particular Watson-Crick base pairs.

[0080] As used herein, a nucleic acid that is "substantially complementary" or "partially complementary" to at least a portion of messenger RNA (mRNA) refers to a nucleic acid that is substantially or partially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding a gene). In certain embodiments, the continuous portion of the mRNA is one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 4 to 17. For example, a nucleic acid is complementary to at least a portion of the mRNA of a gene if its sequence is substantially or partially complementary to an uninterrupted portion of the mRNA of the gene of interest.

[0081] Thus, in some preferred embodiments, the antisense oligonucleosides disclosed herein are completely complementary to the target gene sequence.

[0082] In other embodiments, the antisense oligonucleosides disclosed herein are substantially or partially complementary to the target RNA sequence and include a continuous 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 equivalent region of the target RNA sequence over its entire length.

[0083] In some embodiments, the nucleic acids of the invention, such as siRNAs, comprise a sense strand that is substantially or partially complementary to an antisense oligonucleotide, which in turn is complementary to a target gene sequence and comprises a contiguous 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 the antisense strand 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] In some embodiments, the nucleic acids of the invention, such as siRNAs, comprise an antisense strand that is substantially or partially complementary to a target sequence and 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, and comprises a contiguous nucleoside sequence.

[0085] As used herein, a "subject" is an animal, e.g., a mammal, e.g., a primate (e.g., human, non-human primate, e.g., monkey and chimpanzee), or a non-primate or bird, that endogenously or heterologously expresses a target gene when the target gene sequence has sufficient complementarity with a nucleic acid, e.g., an siRNA agent, to promote target knockdown. In certain preferred embodiments, the subject is a human.

[0086] The term "treating" or "treatment" refers to beneficial or desired results, including, but not limited to, alleviation or amelioration of one or more symptoms related to gene expression. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. Treatment can include prevention of the occurrence of complications, e.g., reduction of liver damage in a subject having a liver infection.

[0087] As used herein, "therapeutically effective amount" is intended to include an amount of a nucleic acid, such as siRNA, that is sufficient to effect the treatment of a subject having a disease, i.e., to reduce, ameliorate, or maintain the treatment of the disease (e.g., by reducing, improving, or maintaining an existing disease or one or more symptoms of the disease or its associated complications) when administered to a patient.

[0088] As used herein, the phrase "pharmaceutically acceptable" refers 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 problems or complications commensurate with a reasonable benefit / risk ratio.

[0089] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ or portion 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 to be treated.

[0090] It is intended that where a value or range of values of a parameter is recited, values intermediate to the recited values and ranges are also intended to be part of the present invention.

[0091] As used herein, the articles "a" and "an" are used to refer to one or more (i.e., at least one) of the grammatical objects of the article.

[0092] The term "including" is used herein to mean the phrase "including but not limited to" and is used interchangeably therewith.

[0093] The term "or" is used herein to mean the term "and / or" and is used interchangeably therewith, unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand or sense strand and antisense strand".

[0094] The term "about" is used herein to mean within a typical tolerance range in the art. For example, "about" can be understood to be about two standard deviations from the average. In certain embodiments, "about" means +10%. In certain embodiments, "about" means +5%. When "about" is present before a series of numbers or a range, it is understood that "about" can modify each of the numbers in the series or range.

[0095] The term "at least" before a number or a 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 apparent from the context. For example, the number of nucleosides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides out of 21 nucleoside nucleic acid molecules" means that 18, 19, 20, or 21 nucleosides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0096] As used herein, "not exceeding" or "less than" is understood to be from the logical lower value or integer to zero, adjacent to the value in the phrase and as logical in the context. For example, a duplex having an overhang of "not exceeding 2 nucleosides" has an overhang of 2, 1, or 0 nucleosides. When "not exceeding" is present before a series of numbers or a range, it is understood that "not exceeding" can modify each of the numbers in the series or range.

[0097] The terminal region of the strand is the last 5 nucleosides from the 5' or 3' end.

[0098] The various embodiments of the present invention can be combined as determined to be appropriate by those skilled in the art.

[0099] Abasic nucleoside In certain embodiments, one, such as two, such as three, such as four, or more abasic nucleosides are present in the nucleic acid according to the present invention. Abasic nucleosides are modified nucleosides because they lack the base normally found at the 1-position of the sugar moiety. Typically, hydrogen is present at the 1-position of the sugar moiety of the abasic nucleosides present in the nucleic acid according to the present invention.

[0100] The abasic nucleoside is in the terminal region of the second strand and is preferably located within the terminal 5 nucleosides at the end of the strand. The terminal region may be the terminal 5 nucleosides including the abasic nucleoside.

[0101] The second strand may include the following as preferred features (all of these 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 the 5'-terminal region of the second strand, and / or Two or more abasic nucleosides in the 5'-terminal region of the second strand, wherein the abasic nucleosides are present in the overhangs described herein, and / or Two or more consecutive abasic nucleosides in the terminal region of the second strand, preferably with one of the abasic nucleosides being the terminal nucleoside, and / or Two or more consecutive abasic nucleosides in the 5'-terminal region of the second strand, preferably with one of the abasic nucleosides being the terminal nucleoside in the 5'-terminal region of the second strand, and / or An inverted internucleoside linkage connects at least one abasic nucleoside to an adjacent base nucleoside in the terminal region of the second strand, and / or In the 5'-terminal region of the second strand, the reverse nucleoside linkage connects at least one abasic nucleoside to an adjacent base nucleoside, and / or The abasic nucleoside as the penultimate nucleoside is connected to a nucleoside that is not the terminal nucleoside (referred to herein as the third last nucleoside) via a reverse linkage, and / or When the strand is read in the direction towards the end including the terminal nucleoside, the abasic nucleoside as two terminal nucleosides connected via a 5'-3' linkage, When the strand is read in the direction towards the end including the terminal nucleoside, the abasic nucleoside as two terminal nucleosides connected via a 3'-5' linkage, The abasic nucleoside as the two terminal positions, wherein the penultimate nucleoside is connected to the third last nucleoside via a reverse linkage, and the reverse linkage is a 5-5' reverse linkage or a 3'-3' reverse linkage, The abasic nucleoside as the two terminal positions, wherein the penultimate nucleoside is connected to the third last nucleoside via a reverse linkage, and is any of the following: (1) The reverse linkage is a 5-5' reverse linkage, and the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'5' when read towards the end including the terminal abasic nucleoside and the penultimate abasic nucleoside, or (2) The reverse linkage is a 3-3' reverse linkage, and the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 5'3' when read towards the end including the terminal abasic nucleoside and the penultimate abasic nucleoside.

[0102] Preferably, an abasic nucleoside is present at the end of the second strand.

[0103] Preferably, two or at least two abasic nucleosides are present in the terminal region of the second strand, preferably at the end and the penultimate position.

[0104] Preferably, two or more abasic nucleosides are consecutive, for example, all abasic nucleosides may be consecutive. For example, one or two or three or four nucleosides at the end may be abasic nucleosides.

[0105] The abasic nucleoside may also be linked to the adjacent nucleoside through a 5'-3' phosphodiester linkage or a reverse linkage, except when there is only one abasic nucleoside at the end. When there is only one abasic nucleoside at the end, it has a reverse linkage to the adjacent nucleoside.

[0106] Reverse linkage (which may also be called inverted linkage and is also found in the art) includes any of 5'-5', 3'-3', 3'-2' or 2'-3' phosphodiester linkages between adjacent sugar moieties of nucleosides.

[0107] Non-terminal abasic nucleosides each have two phosphodiester bonds with their respective adjacent nucleosides, and these may be reverse linkages, or 5'-3 phosphodiester linkages, or one each.

[0108] A preferred embodiment includes two abasic nucleosides at the end and the second last position of the second strand, and the reverse nucleoside internucleoside linkage is located between the second last (abasic) nucleoside and the third last nucleoside.

[0109] Preferably, two abasic nucleosides are present at the end and the second last position of the second strand, the second last nucleoside is linked to the third last nucleoside through a reverse nucleoside internucleoside linkage, and is linked to the terminal nucleoside through a 5'-3' or 3'-5' phosphodiester linkage (read in the direction of the end of the molecule).

[0110] Preferably, the nucleic acid according to the present invention contains one or more abasic nucleosides, and optionally, one or more abasic nucleosides are in the terminal region of the second strand and / or at least one abasic nucleoside is linked to an adjacent base nucleoside through a reverse nucleoside internucleoside linkage.

[0111] Typically, the second strand contains two consecutive abasic nucleosides in the 5'-terminal region of the second strand, wherein one of the abasic nucleosides is the terminal nucleoside in the 5'-terminal region of the second strand and the other abasic nucleoside is the second last nucleoside in the 5'-terminal region of the second strand, (a) the second last abasic nucleoside is connected to the adjacent first base nucleoside in the adjacent 5'-terminal proximal region through a reverse nucleoside internucleoside linkage, (b) the reverse linkage is a 5-5' reverse linkage, and (c) the linkage between the terminal abasic nucleoside and the second last abasic nucleoside is 3'5' when read towards the end containing the terminal abasic nucleoside and the second last 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, the first phosphorothioate internucleoside linkage is between the adjacent first base nucleoside in the 5'-terminal proximal region of the second strand as in (a) and the adjacent second base nucleoside, and the second phosphorothioate internucleoside linkage is between the adjacent second base nucleoside and the adjacent third base nucleoside in the 5'-terminal proximal region of the second strand, (iii) two phosphorothioate internucleoside linkages are respectively between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in the 5'-terminal region and the 3'-terminal region of the first strand are respectively attached to their 5' and 3' adjacent second last nucleosides by phosphorothioate internucleoside linkages, and each of the first 5' and 3' second last nucleosides is attached to their 5' and 3' adjacent third last nucleosides by phosphorothioate internucleoside linkages, (iv) 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.

[0112] Examples of structures are as follows (the specific RNA nucleosides shown are not limiting and can be any RNA nucleoside):

[0113] A 3'-3' inverted bond (also showing the 5'-3' direction of the last phosphodiester bond between two abasic molecules, reading towards the end of the molecule)

[0114]

Chem.

[0115] Exemplify a 5'-5' inverted bond (also showing the 3'-5' direction of the last phosphodiester bond between two abasic molecules, reading towards the end of the molecule)

[0116]

Chem.

[0117] The abasic nucleoside(s) present in the nucleic acid are provided in the presence of an inverted nucleoside linkage(s), i.e., a 5'-5' or 3'-3' inverted nucleoside linkage. The inverted linkage results from a change in the orientation of adjacent nucleoside sugars such that the sugar has a 3'-5' orientation as opposed to the conventional 5'-3' orientation (referring to the numbering of the ring atoms on the nucleoside sugar). The abasic nucleoside(s) present in the nucleic acid of the present invention preferably contain such inverted nucleoside sugars.

[0118] In the case of a terminal nucleoside having an inverted orientation, this results in a stereoconfiguration of the "inverted" terminus for the overall nucleic acid. Certain structures shown and referred to herein are represented using the conventional 5'-3' direction (with reference to the numbering of the ring atoms on the nucleoside sugar), but the change in orientation and the presence of a terminal nucleoside having a proximal 3'-3' reverse linkage results in a nucleic acid having an overall 5'-5' terminus structure (i.e., the conventional 3' terminal nucleoside becomes the 5' terminal nucleoside). Alternatively, it is understood that the change in orientation and the presence of a terminal nucleoside having a proximal 5'-5' reverse linkage results in a nucleic acid having an overall 3'-3' terminus structure.

[0119] The proximal 3'-3' or 5'-5' reverse linkages described herein can include reverse linkages that are directly adjacent / 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' reverse linkages described herein can include reverse linkages that are adjacent to two or more nucleosides having an inverted orientation, e.g., two or more terminal region nucleosides having an inverted orientation, e.g., a terminal nucleoside and the penultimate nucleoside. In this way, the reverse linkage can be attached to the penultimate nucleoside having an inverted orientation. One of ordinary skill in the art will understand that the above-described inverted orientation can result in a nucleic acid molecule having an overall 3'-3' or 5'-5' terminus structure as described herein, but that the presence of one or more additional reverse linkages and / or nucleosides having an inverted orientation can also result in the overall nucleic acid having a 3'-5' terminus structure corresponding to the conventionally arranged 5' / 3' terminus.

[0120] In one aspect, the nucleic acid may have a 3'-3' reverse linkage, and the terminal sugar moiety may contain a 5'OH rather than a 5' phosphate group at the 5' position of the terminal sugar.

[0121] Thus, one of ordinary skill in the art will clearly understand that when reverse linkage(s) exist, 5'-5', 3'-3', and 3'-5' (read in the direction of their termini) terminal variants of the conventional 5'-3' structure (referring to the numbering of the ring atoms on the terminal nucleoside sugar) as shown herein are within the scope of the present disclosure.

[0122] For example, in the situation of one or more nucleosides having a reverse orientation that results in reverse nucleoside linkages and / or inverted termini, and also where the relative position of a 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' terminus of the nucleic acid, the 5' or 3' terminus is the conventional 5' or 3' terminus that would have been present if the reverse linkage were not in a fixed position, and the conventional 5' or 3' terminus is determined by considering the directionality of the majority of the internal nucleoside linkages and / or the nucleoside orientation within the nucleic acid. From these internal linkages and / or nucleoside orientations, it is possible to identify which terminus of the nucleic acid would constitute the conventional 5' and 3' termini of a molecule without reverse linkages (referring to the numbering of the ring atoms on the terminal nucleoside sugar).

[0123] For example, in the structure shown below, abasic residues are present at the first two positions located at the 5' terminus. When the terminal nucleoside has an inverted orientation, the 5' terminus shown in the following schematic, which is the conventional 5' terminus, may actually contain a 3'OH considering the inverted nucleoside at the terminal position. Nevertheless, the majority of the molecule contains conventional nucleoside linkages that run from the 3'OH of one sugar to the 5' phosphate of the next sugar when read in the standard 5'[PO4] to 3'[OH] direction of the nucleic acid molecule (referring to the numbering of the ring atoms on the nucleoside sugar), and this can be used to determine the conventional 5' and 3' termini that would be seen in the absence of the stereochemistry of the inverted terminus.

[0124] 5'A - A - Me - Me - Me - Me - Me - Me - Me - Me - F - F - F - Me - Me - Me - Me - Me - Me - Me - Me 3'

[0125] The reverse linkage is preferably located at the end of a nucleic acid, such as RNA, which is distal to the ligand portion of the molecule, such as the GalNAc-containing portion.

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

[0127] In certain embodiments, the 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 wherein the nucleic acid for inhibiting the expression of the target gene comprises 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 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 through a reverse nucleoside internucleoside linkage to the adjacent first base nucleoside in the adjacent 5'-terminal proximal region, (b) the reverse linkage is a 5-5' reverse linkage, (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when read towards the end comprising the terminal abasic nucleoside and the penultimate abasic nucleoside, relates to a nucleic acid.

[0128] In certain embodiments, the 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 wherein the nucleic acid for inhibiting the expression of the target gene comprises 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 contains 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 second last nucleoside in the 5'-terminal region of the second strand, (a) the second last abasic nucleoside is connected through a reverse nucleoside internucleoside linkage to the adjacent first base nucleoside in the adjacent 5'-terminal proximal region, (b) the reverse linkage is a 5-5' reverse linkage, (c) the linkage between the terminal abasic nucleoside and the second last abasic nucleoside is 3'-5' when read towards the end including the terminal abasic nucleoside and the second last abasic nucleoside, (iii) two phosphorothioate nucleoside internucleoside linkages are each present between three consecutive positions in the 5'-terminal proximal region of the second strand, the first phosphorothioate nucleoside 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 the second phosphorothioate nucleoside 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 nucleoside internucleoside linkages are each present between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in the 5'-terminal region and the 3'-terminal region of the first strand are each attached by a phosphorothioate nucleoside internucleoside linkage to the respective 5' and 3' adjacent second last nucleosides, and the respective 5' and 3' second last nucleosides are attached by a phosphorothioate nucleoside internucleoside linkage to the respective 5' and 3' adjacent third last nucleosides, (v) 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. Relates to a nucleic acid.

[0129] In certain embodiments, the present invention 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 the expression of a target gene, comprising a double-stranded region containing The second strand has the following 5'-terminal motif

[0130] [Chemical formula]

[0131] [wherein B represents a nucleoside base, T represents H, OH or a 2'-ribose modification, Z represents the remaining nucleosides of the second strand] Relates to a nucleic acid containing two consecutive abasic nucleosides in the 5'-terminal region of the second strand, present as

[0132] In certain embodiments, the present invention 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 the expression of a target gene, comprising a double-stranded region containing The second strand has the following 5'-terminal motif

[0133] [Chemical formula]

[0134] [wherein B represents a nucleoside base, T represents H, OH, or a 2'-ribose modification, V represents O or S (preferably O), R represents H or C 1~4 alkyl (preferably H), Z represents the remaining nucleosides of the second strand], more preferably the following 5'-terminal motif

[0135]

Chemical formula

[0136] [wherein, B represents a nucleoside base, T represents H, OH, or a 2'-ribose modification, Z represents the remaining nucleosides of the second strand] 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 invention provides a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand comprising a double-stranded region for inhibiting the expression of a target gene, wherein the second strand has the following 5'-terminal motif

[0138]

Chemical formula

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

[0140]

Chemical formula

[0141] [wherein, B represents a nucleoside base, T represents H, OH or a 2'-ribose modification, Z contains 11 to 26 consecutive nucleosides, preferably 15 to 21 consecutive nucleosides, more preferably 19 consecutive nucleosides] and contains two consecutive abasic nucleosides in the 5'-end region of the second strand, which exists as

[0142] In some embodiments, the modification pattern of the second (sense) strand of the nucleic acid according to the present invention is ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me [wherein, ia represents an inverted abasic nucleoside] and comprises or consists of.

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

[0144]

Chemical formula

[0145] [wherein, B represents the nucleoside base of the first base nucleoside in the 5'-end region of the second strand, T represents a 2'-Me ribose modification, Z represents the remaining consecutive base nucleosides of the second strand] and comprises.

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

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

[0148]

Chemical formula

[0149] [where 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 represents H or C 1~4 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]

Chemical formula

[0151] [where 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 19 consecutive base nucleosides of the remaining of the second strand including.

[0152] In a preferred embodiment, the modification pattern of the second (sense) strand of the nucleic acid according to the invention is 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 [wherein, (s) is a phosphorothioate nucleoside internucleoside linkage, and ia represents an inverted abasic nucleoside] comprises or consists of.

[0153] In such an embodiment, the second strand preferably has the following 5'-terminal motif

[0154]

Chemical formula

[0155] [wherein 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 represents H or C 1~4 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'-terminal motif

[0156]

Chemical formula

[0157] [wherein 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 base nucleosides of the second strand] and includes.

[0158] Length of nucleic acid In one aspect, i) the first strand of the nucleic acid has a length 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 of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

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

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

[0161] In other preferred embodiments, the nucleic acids of the invention, such as RNA, such as 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] The nucleic acids characterized by the present 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, NY, USA, which is incorporated herein by reference.

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

[0165] Specific examples of nucleic acids, such as siRNA compounds useful in the embodiments described herein, include, but are not limited to, RNAs containing a modified backbone or RNAs lacking natural internucleoside linkages. Nucleic acids such as RNAs having a modified backbone include, inter alia, those having no phosphorus atoms in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified nucleic acids, such as RNAs, that have no phosphorus atoms in their internucleoside backbone can also be considered oligonucleosides. In some embodiments, a modified nucleic acid, such as an siRNA, has a phosphorus atom in its internucleoside backbone.

[0166] Modified nucleic acids, such as RNA backbones, include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates, their 2'-5' linkage analogs, and those having inverted polarity with adjacent pairs of nucleoside units linked 5'-3' or 5'-2'. Also included are various salts, mixed salts, and free acid forms.

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

[0168] In certain preferred embodiments, the nucleic acid contains 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 of the sense strand and all of the nucleosides of the antisense strand contain modifications.

[0171] In some embodiments, all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand contain modifications.

[0172] In some embodiments, all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand include modifications.

[0173] In one embodiment, at least one of the modified nucleosides is selected from the group consisting of deoxy-nucleoside, 3'-terminal deoxy-thymidine (dT) nucleoside, 2'-O-methyl modified nucleoside (also referred to herein as 2'-Me, where Me is methoxy), 2'-fluoro modified nucleoside, 2'-deoxy-modified nucleoside, locked nucleoside, unlocked nucleoside, nucleoside with restricted conformation, constrained ethyl nucleoside, abasic nucleoside, 2'-amino-modified nucleoside, 2'-O-allyl-modified nucleoside, 2'-O-alkyl-modified nucleoside, 2'-hydroxyl-modified nucleoside, 2'-methoxyethyl modified nucleoside, 2'-O-alkyl-modified nucleoside, morpholino nucleoside, phosphoramidate, nucleoside containing unnatural base, tetrahydropyran modified nucleoside, 1,5-anhydrohexitol modified nucleoside, cyclohexenyl modified nucleoside, nucleoside containing phosphorothioate group, nucleoside containing methylphosphonate group, nucleoside containing 5'-phosphate, and nucleoside containing 5'-phosphate mimetic. In another embodiment, the modified nucleoside includes a short sequence of 3'-terminal deoxy-thymidine nucleoside (dT).

[0174] The modification on the nucleoside can 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 2'-Me or 2'-F modifications.

[0176] Preferred nucleic acids contain 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 a modification at the 2'-OH group of the ribose sugar, optionally selected from 2'-Me or 2'-F modifications.

[0178] A nucleic acid wherein each of the first and second strands contains 2'-Me and 2'-F modifications.

[0179] A nucleic acid wherein the first strand contains a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications.

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

[0182] The first strand is as follows (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 [wherein X1 is a thermolabile modification] and contains a 2'-sugar modification pattern of a nucleic acid.

[0183] A nucleic acid wherein the first strand contains a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 5 2'-F modifications.

[0184] The first strand is as follows (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 [wherein, X1 is a heat destabilizing modification] A nucleic acid comprising a 2'-sugar modification pattern of

[0185] A nucleic acid in which the first strand comprises 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 strand consists of 7 2'-F modifications.

[0186] Preferably, a nucleic acid comprising at least one heat destabilizing modification at one or more of positions 1 to 9 of the first strand counted from the 1st position 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 destabilizing modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid, more preferably (S)-glycol nucleic acid.

[0187] The nucleic acid described herein, wherein the first strand comprises at least one duplex heat destabilizing modification within the first 9 nucleoside positions of its 5'-region.

[0188] A nucleic acid comprising at least one duplex heat destabilizing modification within nucleoside positions 2 to 9 of the 5'-region of the first strand.

[0189] A nucleic acid comprising at least one duplex heat destabilizing modification within nucleoside positions 2 to 8 of the 5'-region of the first strand.

[0190] A nucleic acid comprising at least one duplex heat destabilizing modification within nucleoside positions 3 to 8 of the 5'-region of the first strand.

[0191] A nucleic acid comprising at least one duplex heat destabilizing modification within nucleoside positions 4 to 8 of the 5'-region of the first strand.

[0192] A nucleic acid comprising at least one duplex heat destabilizing modification within nucleoside position 6 or 7 of the 5'-region of the first strand.

[0193] A nucleic acid comprising at least one duplex thermolability modification at the 6th position of the first strand when counted from the 1st position of the first strand.

[0194] The nucleic acid described herein, wherein the destabilizing modification comprises at least one glycol nucleic acid (GNA), more preferably (S)-glycol nucleic acid.

[0195] Glycol nucleic acid (GNA), sometimes also called glycerol nucleic acid, is a nucleic acid that is similar to DNA or RNA but has a different composition of its sugar-phosphate backbone, using propylene glycol instead of ribose or deoxyribose. A single GNA nucleotide can form Watson-Crick base pairs with (deoxy)ribonucleotides but is very unstable when incorporated into a DNA or RNA duplex.

[0196] The nucleic acid described herein, wherein the destabilizing modification comprises at least one unlocked nucleic acid (UNA).

[0197] The term "UNA" refers to an unlocked acyclic nucleic acid in which all sugar linkages have been removed to form an "unlocked" sugar residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' (i.e., the carbon-oxygen-carbon covalent bond between the C1' carbon and the C4' carbon) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' carbon and the C3' carbon) has been removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0198] The second strand is as follows (5'-3'): (Me)8-(F)3-(Me) 10 A nucleic acid comprising a 2'-sugar modification pattern of

[0199] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 Comprising a 2'-sugar modification pattern of, and the first strand comprises at least one double-stranded thermal destabilizing modification within the first 9 nucleoside positions of its 5' region.

[0200] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 Comprising a 2'-sugar modification pattern of, and the first strand comprises at least one double-stranded thermal destabilizing modification within the first 9 nucleoside positions of its 5' region, The nucleosides of the first strand comprise a 2'-sugar modification pattern, 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 strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise 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 strand consists of 3, 5 or 7 2'-F modifications.

[0201] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 comprising a 2'-sugar modification pattern of, wherein the first strand comprises at least one double-stranded thermal destabilizing modification within positions 2 to 9 of the nucleosides in the 5'-region of the first strand, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0202] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 comprising a 2'-sugar modification pattern of, wherein the first strand comprises at least one double-stranded thermal destabilizing modification within positions 2 to 8 of the nucleosides in the 5'-region of the first strand, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0203] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 including a 2'-sugar modification pattern of, and the first strand includes at least one double-strand thermally destabilizing modification within positions 3 to 8 of the nucleosides in the 5'-region of the first strand, the nucleosides of the first strand include a 2'-sugar modification pattern, 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 strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand include 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 strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0204] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 including a 2'-sugar modification pattern of, and the first strand includes at least one double-strand thermally destabilizing modification within positions 4 to 8 of the nucleosides in the 5'-region of the first strand, The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. A nucleic acid.

[0205] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 include a 2'-sugar modification pattern, and the first strand includes at least one double-strand thermal destabilizing modification within positions 6 or 7 of the nucleosides in the 5'-region of the first strand. The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. A nucleic acid.

[0206] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 comprising a 2'-sugar modification pattern, wherein the first strand comprises at least one duplex thermal destabilizing modification at position 6 in the 5'-region of the first strand, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0207] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 comprising a 2'-sugar modification pattern, wherein the first strand comprises at least one duplex thermal destabilizing modification at position 7 in the 5'-region of the first strand, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0208] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 comprising a 2'-sugar modification pattern, wherein the first strand is as follows (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 [wherein X1 is a heat-labile modification] A nucleic acid comprising a 2'-sugar modification pattern of

[0209] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): (Me)8-(F)3-(Me) 10 comprising a 2'-sugar modification pattern, wherein the first strand is as follows (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 [wherein X1 is a heat-labile modification] A nucleic acid comprising a 2'-sugar modification pattern of

[0210] The second strand is as follows (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 [wherein ia represents an inverted abasic nucleoside] A nucleic acid comprising a 2'-sugar modification pattern of

[0211] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] A nucleic acid comprising a 2'-sugar and a non-nucleobase modification pattern, wherein the first strand comprises at least one duplex thermolabilizing modification within the first 9 nucleoside positions of its 5'-region.

[0212] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 having a length of at least 17 nucleosides, 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 non-nucleobase nucleoside] Comprising a 2'-sugar and a non-nucleobase modification pattern, wherein the first strand comprises at least one duplex thermolabilizing modification within the first 9 nucleoside positions of its 5'-region, The nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications.

[0213] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 having a length of at least 17 nucleosides, 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 non-nucleobase nucleoside] Comprising a 2'-sugar and a non-nucleobase modification pattern, wherein the first strand comprises at least one duplex thermolabilizing modification within nucleoside positions 2 to 9 of the 5'-region of the first strand, The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. A nucleic acid.

[0214] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including a 2'-sugar and abasic modification pattern of, the first strand including at least one double-stranded thermal destabilizing modification within positions 2 to 8 of the nucleosides in the 5'-region of the first strand, The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. A nucleic acid.

[0215] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] comprising a 2'-sugar and abasic modification pattern, wherein the first strand comprises at least one duplex thermal destabilizing modification within nucleosides 3 to 8 of the 5'-region of the first strand, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0216] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 having a length of at least 17 nucleosides, 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] comprising a 2'-sugar and abasic modification pattern, wherein the first strand comprises at least one duplex thermal destabilizing modification within nucleosides 4 to 8 of the 5'-region of the first strand, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0217] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] comprising the 2'-sugar and abasic modification patterns of, and the first strand comprises at least one double-stranded thermally destabilizing modification within positions 6 or 7 of the nucleosides in the 5'-region of the first strand, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications, in particular, the nucleosides of the first strand comprise a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications, a nucleic acid.

[0218] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] comprising the 2'-sugar and abasic modification patterns of, and the first strand comprises at least one double-stranded thermally destabilizing modification at position 6 in the 5'-region of the first strand, The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. A nucleic acid.

[0219] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including a 2'-sugar and abasic modification pattern of, the first strand including at least one double-stranded thermal destabilizing modification at the 7-position of the 5'-region of the first strand, The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. A nucleic acid.

[0220] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] comprising a 2'-sugar and a nucleobase-free modification pattern, wherein the first strand is as follows (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 [wherein X1 is a heat-labile modification] A nucleic acid comprising a 2'-sugar modification pattern of.

[0221] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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 nucleobase-free nucleoside] comprising a 2'-sugar and a nucleobase-free modification pattern, wherein the first strand is as follows (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 [wherein X1 is a heat-labile modification] A nucleic acid comprising a 2'-sugar modification pattern of.

[0222] In certain embodiments, the nucleic acid, e.g., an siRNA agent, further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage. For example, the phosphorothioate or methylphosphonate internucleoside linkage may be at the 3'-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.

[0223] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkage is 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.

[0224] In certain embodiments, phosphorothioate or methylphosphonate nucleoside internucleotide linkages are at both or either the 5' and 3' termini or terminal regions of one strand, i.e., the sense or antisense strand, or at the termini of both strands, the sense and antisense strands.

[0225] Any nucleic acid may contain one or more phosphorothioate (PS) modifications within the nucleic acid, e.g., at least two PS internucleoside linkages at the termini of the strand.

[0226] At least one of the oligoribonucleoside strands preferably contains at least two consecutive phosphorothioate modifications at the last three nucleosides of the oligonucleoside.

[0227] Accordingly, the invention also relates to: nucleic acids as disclosed herein that contain phosphorothioate internucleotide linkages between at least two or three consecutive positions, such as in the 5' and / or 3' terminal regions and / or regions near the termini of the second strand, whereby the region near the termini is preferably adjacent to the terminal region in which one or more abasic nucleosides of the second strand are located.

[0228] Nucleic acids as disclosed herein that contain phosphorothioate internucleotide linkages between at least two or three consecutive positions in the 5' and / or 3' terminal regions of the first strand, whereby preferably the terminal positions in the 5' and / or 3' terminal regions of the first strand are attached by phosphorothioate internucleotide linkages to their adjacent positions.

[0229] The nucleic acid strand may be an RNA that contains a phosphorothioate internucleotide linkage between three nucleosides adjacent to an abasic nucleoside located at both termini.

[0230] In a preferred embodiment, the present invention relates to a nucleic acid in which two phosphorothioate internucleoside linkages are respectively present between three consecutive positions in the region near the 5'-end of the second strand, the first phosphorothioate internucleoside linkage is present between the first base nucleoside and the adjacent second base nucleoside when read from the 5'-end in the region near the 5'-end of the second strand, and the second phosphorothioate internucleoside linkage is present between the second base nucleoside and the adjacent third base nucleoside in the region near the 5'-end of the second strand.

[0231] In a more preferred embodiment, the present invention relates to a nucleic acid in which two phosphorothioate internucleoside linkages are respectively present between three consecutive positions in both the 5'-end region and the 3'-end region of the first strand, whereby the terminal nucleosides in the 5'-end region and the 3'-end region of the first strand are respectively attached to the 5'- and 3'-adjacent second-last nucleosides by phosphorothioate internucleoside linkages, and the 5'- and 3'-second-last nucleosides are respectively attached to the 5'- and 3'-adjacent third-last nucleosides by phosphorothioate internucleoside linkages.

[0232] The second strand is 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] A nucleic acid comprising the 2'-sugar and abasic modification pattern thereof.

[0233] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including the 2'-sugar and abasic modification pattern of , wherein the first strand includes at least one duplex heat destabilizing modification within the first 9 nucleoside positions of its 5'-region, preferably, a phosphorothioate internucleoside linkage is present between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in the 5'-terminal region and the 3'-terminal region of the first strand are each attached to their respective 5'- and 3'-adjacent penultimate nucleosides by a phosphorothioate internucleoside linkage, and the respective 5'- and 3'-penultimate nucleosides are attached to their respective 5'- and 3'-adjacent antepenultimate nucleosides by a phosphorothioate internucleoside linkage, a nucleic acid.

[0234] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 that is 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] including the 2'-sugar and abasic modification pattern of , wherein the first strand includes at least one duplex heat destabilizing modification within the first 9 nucleoside positions of its 5'-region, The nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, two phosphorothioate internucleoside linkages are present respectively between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in each of the 5'-terminal region and the 3'-terminal region of the first strand are attached respectively to the 5'- and 3'-adjacent second last nucleosides by the phosphorothioate internucleoside linkages, and the 5'- and 3'-second last nucleosides are attached respectively to the 5'- and 3'-adjacent third last nucleosides by the phosphorothioate internucleoside linkages, nucleic acid.

[0235] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including the 2'-sugar and abasic modification pattern of, and the first strand includes at least one double-stranded thermal destabilizing modification within positions 2 to 9 of the nucleosides in the 5'-region of the first strand. The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, 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, whereby the terminal nucleosides in the 5'-terminal region and the 3'-terminal region of the first strand are each attached to their respective 5'- and 3'-adjacent penultimate nucleosides by phosphorothioate internucleoside linkages, and the respective 5'- and 3'-penultimate nucleosides are attached to their respective 5'- and 3'-adjacent antepenultimate nucleosides by phosphorothioate internucleoside linkages, a nucleic acid.

[0236] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including a 2'-sugar and abasic modification pattern of, and the first strand includes at least one double-stranded thermolability modification within positions 2 to 8 of the nucleosides in the 5'-region of the first strand. The nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, two phosphorothioate internucleoside linkages are present respectively between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in each of the 5'-terminal region and the 3'-terminal region of the first strand are attached respectively to the 5'- and 3'-adjacent second last nucleosides by the phosphorothioate internucleoside linkages, and the 5'- and 3'-second last nucleosides are attached respectively to the 5'- and 3'-adjacent third last nucleosides by the phosphorothioate internucleoside linkages, nucleic acid.

[0237] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] comprising the 2'-sugar and abasic modification pattern of, and the first strand contains at least one double-stranded thermal destabilizing modification within positions 3 to 8 of the nucleosides in the 5'-region of the first strand. The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, two phosphorothioate internucleoside linkages are present respectively between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in the 5'-terminal region and the 3'-terminal region of the first strand are each attached by a phosphorothioate internucleoside linkage to the respective 5'- and 3'-adjacent second last nucleosides, and the respective 5'- and 3'-second last nucleosides are attached by a phosphorothioate internucleoside linkage to the respective 5'- and 3'-adjacent third last nucleosides, nucleic acid.

[0238] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including a 2'-sugar and abasic modification pattern of, and the first strand includes at least one double-stranded thermal destabilizing modification within positions 4 to 8 of the nucleosides in the 5'-region of the first strand. The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, two phosphorothioate internucleoside linkages are present respectively between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in the 5'-terminal region and the 3'-terminal region of the first strand are each attached by a phosphorothioate internucleoside linkage to the respective 5'- and 3'-adjacent second-to-last nucleosides, and the respective 5'- and 3'-second-to-last nucleosides are attached by a phosphorothioate internucleoside linkage to the respective 5'- and 3'-adjacent third-to-last nucleosides, nucleic acid.

[0239] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including the 2'-sugar and abasic modification pattern of, and the first strand includes at least one double-stranded thermal destabilizing modification within positions 6 or 7 of the nucleosides in the 5'-region of the first strand. The nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, 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, whereby the terminal nucleosides in each of the 5'-terminal region and the 3'-terminal region of the first strand are attached to their respective 5'- and 3'-adjacent second-to-last nucleosides by the phosphorothioate internucleoside linkages, and the second-to-last nucleosides of each 5'- and 3'-are attached to their respective 5'- and 3'-adjacent third-to-last nucleosides by the phosphorothioate internucleoside linkages, a nucleic acid.

[0240] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] including the 2'-sugar and abasic modification pattern of, and the first strand includes at least one double-stranded thermolability modification at the 6-position of the 5'-region of the first strand. The nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand contain a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, two phosphorothioate internucleoside linkages are present respectively between three consecutive positions in both the 5'-terminal region and the 3'-terminal region of the first strand, whereby the terminal nucleosides in each of the 5'-terminal region and the 3'-terminal region of the first strand are attached respectively to the 5'- and 3'-adjacent second last nucleosides by the phosphorothioate internucleoside linkages, and the second last nucleosides at 5' and 3' are attached respectively to the 5'- and 3'-adjacent third last nucleosides by the phosphorothioate internucleoside linkages, nucleic acid.

[0241] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] contain a 2'-sugar and abasic modification pattern of, and the first strand contains at least one double-stranded thermal destabilizing modification at the 7th position of the 5'-region of the first strand. The nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is not composed of 4 or 6 2'-F modifications. In particular, the nucleosides of the first strand include a 2'-sugar modification pattern, the modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5 or 7 2'-F modifications. Preferably, 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, whereby the terminal nucleosides in each of the 5'-terminal region and the 3'-terminal region of the first strand are attached to their respective 5'- and 3'-adjacent penultimate nucleosides by phosphorothioate internucleoside linkages, and each of the 5'- and 3'-penultimate nucleosides is attached to its respective 5'- and 3'-adjacent antepenultimate nucleoside by a phosphorothioate internucleoside linkage.

[0242] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region that is 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] including a 2'-sugar and abasic modification pattern of, and the first strand is as follows (5'-3'): Me(s)F(s)(Me)3-X1-(Me)7-F-Me-F-(Me)5(s)Me(s)Me [wherein X1 is a thermally destabilizing modification and (s) represents a phosphorothioate linkage] A nucleic acid including a 2'-sugar modification pattern of.

[0243] A nucleic acid comprising a first strand that is at least partially complementary to a portion of 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 double-stranded region having a length of at least 17 nucleosides, 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] and includes a 2'-sugar and abasic modification pattern of, and the first strand is as follows (5'-3'): Me(s)F(s)(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)5(s)Me(s)Me [wherein X1 is a thermally destabilizing modification, and (s) represents a phosphorothioate linkage] and includes a 2'-sugar modification pattern of, nucleic acid.

[0244] The 1-position of the first or second strand is closest to the end of the nucleic acid (ignoring any abasic nucleosides), and with respect to the bond between the sugar moieties of the backbone, reading in the direction away from that end of the molecule, is a nucleoside linked via a 3' to 5' internal bond to an adjacent nucleoside (at the 2-position).

[0245] Thus, the "1-position of the sense strand" can be regarded as the most 5'-side nucleoside (excluding abasic nucleosides) at the conventional 5'-end of the sense strand. Typically, the nucleoside at this 1-position of the sense strand is equivalent to the 5'-nucleoside of the selected target nucleic acid sequence, and more generally, the sense strand has nucleosides equivalent to those of the target nucleic acid sequence starting from this 1-position of the sense strand, allowing for tolerable mismatches between sequences.

[0246] As used herein, "position 1 of the antisense strand" is the most 5'-terminal nucleoside (excluding abasic nucleosides) at the conventional 5'-end of the antisense strand. As described above herein, 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 mentioned above.

[0247] 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 contain phosphorothioate linkages between nucleotides at positions 1-2 and 2-3 of the second strand, reading from position 1 of the second strand.

[0248] Preferred modifications are as follows: Modification pattern 1: 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-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me [wherein X1 is a thermally destabilizing modification], Or modification pattern 2: 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 [wherein X1 is a thermally destabilizing modification].

[0249] Particularly preferred modifications are as follows: Modification pattern 1: 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-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me [wherein, 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-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 [wherein, X1 is a heat destabilizing modification].

[0250] Conjugation Another modification of the nucleic acid of the present invention, for example RNA, for example siRNA, comprises, for example, linking the nucleic acid, for example siRNA, to one or more ligand moieties in order to enhance the activity, cellular distribution, or cellular uptake of the nucleic acid, for example siRNA, for example into cells.

[0251] In some embodiments, the described ligand moiety may be attached to a nucleic acid, for example a siRNA oligonucleoside, via a linker which may or may not be cleavable. The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, for example covalently bonds two parts of a compound.

[0252] The ligand may be attached to the 3' or 5' end of the sense strand.

[0253] The ligand is preferably conjugated to the 3' end of the sense strand of the nucleic acid, for example a siRNA agent.

[0254] Accordingly, in a further aspect, the present invention relates to a conjugate for inhibiting the expression of a target gene in a cell, the conjugate comprising a nucleic acid moiety and one or more ligand moieties, the nucleic acid moiety comprising a nucleic acid disclosed herein.

[0255] In one aspect, 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 the terminal region of the second strand, preferably its 3' terminal region.

[0256] In certain embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative attached to the nucleic acid, e.g., dsiRNA, through a linker.

[0257] Accordingly, the present invention relates to a conjugate wherein the ligand moiety comprises 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 the nucleic acid through a linker The GalNAc ligand may be conjugated directly or indirectly, preferably at the 3' terminal region, to the 5' or 3' terminal region of the second strand of the nucleic acid.

[0258] GalNAc ligands are well known in the art and are described, inter alia, in EP3775207A1.

[0259]

[0260] ​In some embodiments, the GalNAc ligand is included in any one of the linkers shown in FIGS. 1-4 or FIG. 5 (Formula XI), and the "oligonucleotide" may be any nucleic acid disclosed herein. Thus, the "oligonucleotide" may include linkages other than phosphodiester linkages, such as one or more phosphorothioate linkages. Preferably, the nucleic acid according to the invention is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand via a phosphodiester linkage, more preferably to the 3'-terminal region of the second strand.

[0261] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 3, and the "oligonucleotide" may be any nucleic acid disclosed herein. Thus, the "oligonucleotide" may include linkages other than phosphodiester linkages, such as one or more phosphorothioate linkages. Preferably, the nucleic acid according to the invention is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand via a phosphodiester linkage, more preferably to the 3'-terminal region of the second strand.

[0262] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 5 (Formula XI), and the "oligonucleotide" may be any nucleic acid disclosed herein. Thus, the "oligonucleotide" may include linkages other than phosphodiester linkages, such as one or more phosphorothioate linkages. Preferably, the nucleic acid according to the invention is a double-stranded oligonucleoside as defined herein, and the linker is conjugated to the second strand via a phosphodiester linkage, more preferably to the 3'-terminal region of the second strand.

[0263] In some embodiments, the GalNAc ligand is included in any one of the linkers shown in FIGS. 1-4 or FIG. 5 (Formula XI), and the "oligonucleotide" represents the nucleic acid according to the invention, and the 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-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me [wherein, (s) is a phosphorothioate nucleoside internucleoside linkage, and ia represents an inverted abasic nucleoside] comprising a modified second strand having, preferably, the linker conjugated to the 3'-terminal region of the second strand via a phosphodiester bond.

[0264] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 3, "oligonucleotide" represents a nucleic acid according to the present invention, and the nucleic acid according to the present invention has the following modification pattern (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 [wherein, (s) is a phosphorothioate nucleoside internucleoside linkage, and ia represents an inverted abasic nucleoside] comprising a modified second strand having, preferably, the linker conjugated to the 3'-terminal region of the second strand via a phosphodiester bond.

[0265] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 5 (Formula XI), "oligonucleotide" represents a nucleic acid according to the present invention, and the nucleic acid according to the present invention has the following modification pattern (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 [wherein, (s) is a phosphorothioate nucleoside internucleoside linkage, and ia represents an inverted abasic nucleoside] comprising a modified second strand having, preferably, the linker conjugated to the 3'-terminal region of the second strand via a phosphodiester bond.

[0266] In some embodiments, the GalNAc ligand is included in any one of the linkers shown in FIGS. 1-4 or FIG. 5 (Formula XI), and "oligonucleotide" represents the nucleic acid according to the present invention. The nucleic acid according to the present invention has the following modification pattern (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 [wherein, (s) is a phosphorothioate internucleoside linkage, and ia represents an inverted abasic nucleoside] and includes a modified second strand having the following structure:

[0267]

Chemical formula

[0268] [wherein, 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, and Z represents the remaining 19 consecutive base nucleosides of the second strand] has.

[0269] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 3, and "oligonucleotide" represents the nucleic acid according to the present invention. The nucleic acid according to the present invention has the following modification pattern (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 [wherein, (s) is a phosphorothioate internucleoside linkage, and ia represents an inverted abasic nucleoside] and includes a modified second strand having the following structure:

[0270]

Chemical formula

[0271] [wherein, T represents a 2'-Me ribose modification, B represents the nucleoside base of the first two base nucleosides in the 5'-terminal region of the second strand, Z represents the remaining 19 consecutive base nucleosides of the second strand] has.

[0272] In some embodiments, the GalNAc ligand is included in the linker shown in FIG. 5 (Formula XI), and the "oligonucleotide" represents a nucleic acid according to the present invention, and the nucleic acid according to the present invention has the following modification pattern (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 [wherein, (s) is a phosphorothioate internucleoside linkage, and ia represents an inverted abasic nucleoside] includes a modified second strand having, and the second strand has the following structure:

[0273]

Chemical formula

[0274] [wherein, T represents a 2'-Me ribose modification, B represents the nucleoside base of the first two base nucleosides in the 5'-terminal region of the second strand, Z represents the remaining 19 consecutive base nucleosides of the second strand] has.

[0275] Vectors and Cells In one aspect, the present invention provides a cell containing a nucleic acid described herein, such as inhibitory RNA [RNAi].

[0276] In one aspect, the present invention provides a cell containing a vector described herein.

[0277] Pharmaceutically acceptable composition In one aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of a target gene, the pharmaceutical composition comprising a nucleic acid disclosed herein.

[0278] The pharmaceutically acceptable composition may contain excipients and / or carriers.

[0279] 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository 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) buffering agents, 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.

[0280] Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose, etc.), fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or calcium hydrogen phosphate, etc.), lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (such as starch, sodium starch glycolate, etc.), and wetting agents (such as sodium lauryl sulfate, etc.).

[0281] Pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with the nucleic acid and are suitable for non - oral administration can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0282] Formulations for topical administration of nucleic acids can include sterile and non - sterile aqueous solutions, non - aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oil - based vehicles. The solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with the nucleic acid and are suitable for non - oral administration can be used.

[0283] In one embodiment, the nucleic acid or composition is administered in a non-buffered solution. In certain embodiments, the non-buffered solution is saline or water. In other embodiments, the nucleic acid, e.g., an siRNA agent, is administered in a buffered solution. In such embodiments, the buffered solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffered solution can be phosphate buffered saline (PBS).

[0284] Dosage The pharmaceutical composition of the present invention can be administered in a dosage sufficient to inhibit gene expression. Generally, a suitable dosage of the nucleic acid of the present invention, e.g., siRNA, ranges from about 0.001 to about 200.0 milligrams per kilogram of the recipient's body weight per day, generally in the range of about 1 to 50 mg per kilogram of body weight per day. Typically, a suitable dosage of the nucleic acid of the present invention, e.g., siRNA, ranges 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.

[0285] The repeated dosing regimen can include administration of a therapeutic amount of a nucleic acid, e.g., siRNA, at regular intervals such as every other day or once a year. In certain embodiments, the nucleic acid, e.g., siRNA, is administered from about once a month to about once every quarter (i.e., about once every three months).

[0286] In various embodiments, the nucleic acid, such as an 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, such as an siRNA agent, is administered at a dose of about 10 mg / kg to about 30 mg / kg. In certain embodiments, the nucleic acid, such as an 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, such as an agent, is administered at a dose of about 0.1 mg / kg to about 5.0 mg / kg once a week, once a month, once every two months, or quarterly (i.e., once every three months). In certain embodiments, the nucleic acid, such as an siRNA agent, is administered to the subject once a week. In certain embodiments, the nucleic acid, such as an siRNA agent, is administered to the subject once a month. In certain embodiments, the nucleic acid, such as an siRNA agent, is administered quarterly (i.e., every three months).

[0287] After the initial treatment regimen, the treatment can be administered less frequently. For example, after administering weekly or bi-weekly for three months, the administration can be repeated once a month for six months or a year, or longer.

[0288] 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 using delivery through a sustained infusion or controlled release formulation. In that case, the nucleic acid, such as siRNA, contained in each partial dose must correspondingly be smaller in order to achieve the total daily dose. The dosage unit can also be configured for delivery over several days, for example, using a conventional sustained release formulation that provides for the sustained release of the 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 agents at specific sites, such as those that can be used with the agents of the present invention. In this embodiment, the dosage unit contains the corresponding plurality of daily doses.

[0289] In other embodiments, a single dose of the pharmaceutical composition can have long-term persistence such that subsequent doses are administered at intervals not exceeding 3, 4, or 5 days, or at intervals not exceeding 1, 2, 3, or 4 weeks. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered every other month. In certain embodiments, the siRNA is administered from about once a month to about once a quarter (i.e., about once every 3 months), or even every 6 months or 12 months.

[0290] Estimation of the effective dosage and in vivo half-life for an individual nucleic acid, such as siRNA, encompassed by the present invention can be carried out using conventional methods or based on in vivo tests using appropriate animal models as known in the art.

[0291] The pharmaceutical compositions of the present invention can be administered in several ways depending on whether local treatment is desired or systemic treatment is desired, and on the area to be treated. Administration can be local (e.g., by transdermal patch), pulmonary, e.g., by inhalation or insufflation of a powder or aerosol, e.g., by nebulizer, intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration can include intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implant device; or intracranial, e.g., by parenchymal, 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.

[0292] In one embodiment, a nucleic acid, such as an agent, is administered subcutaneously to a subject.

[0293] Nucleic acids, such as siRNA, can be delivered in a manner that targets a particular tissue (e.g., especially in liver cells).

[0294] Method for inhibiting target gene expression The present invention also provides a method for inhibiting the expression of a target gene in a cell. The method comprises contacting the cell with a nucleic acid of the present invention, such as an siRNA agent, such as a double-stranded siRNA agent, in an amount effective to inhibit the expression of the target gene in the cell, thereby inhibiting the expression of the target gene in the cell. It should be noted that the nucleic acid "for inhibiting the expression of the target gene" is preferably a nucleic acid capable of inhibiting target gene expression as described hereinafter in this specification.

[0295] Contacting the cell with a nucleic acid, such as siRNA, such as a double-stranded siRNA agent, may be performed in vitro or in vivo. Contacting the cell with the nucleic acid in vivo includes, for example, contacting a cell or cell population within a subject, such as a human subject, with the nucleic acid, such as siRNA. A combination of in vitro and in vivo methods of contacting the cell is also possible. Contacting the cell may be direct or indirect, as discussed above. Further, contacting the cell may 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, such as a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to the site of interest.

[0296] The term "inhibiting", as used herein, is used interchangeably with "decreasing", "silencing", "down-regulating", "suppressing", and other similar terms, and includes any level of inhibition.

[0297] In some embodiments of the methods of the invention, the expression of the target gene is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or less than the detection level of the assay, when determined by qPCR as described herein and / or when the siRNA is introduced into the target cells by transfection. In certain embodiments, the method comprises a clinically relevant inhibition of the expression of the target gene, as demonstrated by a clinically relevant result, for example, after treatment of a subject with an agent that reduces the expression of the gene.

[0298] In some embodiments, when transfected into cells, the nucleic acids of the invention inhibit the expression of the 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, preferably as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0299] Inhibition of the expression of the target gene can be quantified by the following methods: Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB cell bank) can be maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere. The cells can then be transfected with siRNA duplexes targeting mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 85), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 84)) using 10×3-fold serial dilutions over a final double-strand concentration range of 20 nM to 1 pM. Transfection can be carried out 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 purification can be performed using the RNeasy 96 Kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in a single experiment.

[0300] cDNA synthesis can be carried out using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) can be 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.

[0301] qPCR is performed in duplicate for the cDNA from each well, and the average cycle threshold (Ct) can be calculated. The maximum percent inhibition of target gene expression and the IC50 value can be calculated using the four-parameter (variable slope) model using GraphPad Prism 9.

[0302] Alternatively or additionally, inhibition of target gene expression may be characterized by a decrease in the average relative expression of the target gene.

[0303] In some embodiments, when the cells are transfected with 0.1 nM of the 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.

[0304] In some embodiments, when the cells are transfected with 5 nM of the 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, 0.4 or 0.3, as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0305] The average relative expression 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 Medium (DMEM) supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere. The cells can be transfected with siRNA duplexes targeting mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 85), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 84)) at final duplex concentrations of 5 nM and 0.1 nM. Transfection can be carried out 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 can be 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 can be performed using the RNeasy 96 Kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in two independent experiments.

[0306] cDNA synthesis can be carried out using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) can be 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.

[0307] qPCR is performed in duplicate for the cDNA from each well, and the average Ct can be calculated. Relative target gene expression can be calculated relative to untreated cells, normalized to GAPDH, from the average Ct values using the comparative Ct (ΔΔCt) method.

[0308] Inhibition of target gene expression can be manifested by a decrease in the amount of target gene mRNA as compared to a suitable control.

[0309] In other embodiments, inhibition of target gene expression can be evaluated with respect to a parameter that is functionally related to gene expression, such as a decrease in protein expression or a signaling pathway. Examples of target genes exemplified herein are HCII, ZPI, and B4GALT1.

[0310] Method for treating or preventing diseases related to target gene expression The present invention also provides a method of using a nucleic acid of the present invention, such as siRNA, or a composition containing a nucleic acid of the present invention, such as siRNA, to reduce or inhibit target gene expression in a cell. The method includes contacting the cell with a nucleic acid of the present invention, such as dsiRNA, and maintaining the cell for a time sufficient to obtain degradation of the target mRNA transcript, thereby inhibiting the expression of the target gene in the cell. The decrease in gene expression can be evaluated by any method known in the art.

[0311] In the method of the present invention, the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

[0312] Suitable cells for treatment using the method of the present invention may be any cells that express a target gene related to a disease related to a hemostatic disorder, such as a hemostatic disorder such as hemophilia, especially when the target gene is ZPI or HCII.

[0313] Alternatively, suitable cells for treatment using the method of the present invention may be any cells that express a target gene related to diabetes or cardiovascular disease, especially when the target gene is B4GALT1.

[0314] The in vivo method of the present invention may include administering to a subject a composition containing a nucleic acid of the present invention, such as siRNA, and the nucleic acid, such as siRNA, includes a nucleoside sequence that is complementary to at least a part of the RNA transcript of the target gene of the mammal to be treated.

[0315] The present invention further provides a method for treating a subject in need of treatment. The treatment method of the present invention comprises administering to a subject, such as a subject who would benefit from a decrease or inhibition of the expression of a target gene, a therapeutically effective amount of a pharmaceutical composition containing a nucleic acid such as the siRNA of the present invention, a nucleic acid targeting a gene, or a nucleic acid targeting the target gene.

[0316] The disease to be treated may relate to a hemostatic disorder, such as a disease related to a hemostatic disorder such as hemophilia when the target gene is HCII or ZPI as disclosed herein.

[0317] Haemophilia, or hemophilia, is a mostly genetic hereditary disorder that impairs the body's ability to form blood clots, a process necessary to stop bleeding. This results in longer-term bleeding in the subject after injury, easy bruising, and an increased risk of bleeding within joints or the brain. Subjects with mild disease may only have symptoms after an accident or during surgery. Bleeding into joints, also called arthropathy, can cause permanent damage, while bleeding in the brain can cause long-term headache, seizures, or a decrease in the level of consciousness.

[0318] There are two main types of hemophilia: hemophilia A, which results from low levels of clotting factor VIII, and hemophilia B, which results from low levels of clotting factor IX. They are typically inherited from parents through the X chromosome carrying a non-functional gene. Rarely, new mutations can occur during early development, or hemophilia can develop later in life due to antibodies formed against the clotting factors. Other types include hemophilia C, which results from low levels of factor XI, von Willebrand disease, which results from low levels of a substance called von Willebrand factor, and parahemophilia, which results from low levels of factor V. Hemophilia A, B, and C prevent the proper functioning of the intrinsic pathway; this clotting pathway is needed when there is damage to the endothelium of blood vessels. Acquired hemophilia is associated with cancer, autoimmune disorders, and pregnancy. Diagnosis is by testing blood for its clotting ability and its clotting factor levels.

[0319] In certain embodiments, the nucleic acids of the invention, particularly those that inhibit the expression of ZPI or HCII, are suitable for the treatment, or treatment of, hemophilia A, B, and / or C. In certain embodiments, the nucleic acids of the invention, particularly those that inhibit the expression of ZPI or HCII, are suitable for the treatment, or treatment of, hemophilia A and / or B. In certain embodiments, the nucleic acids of the invention, particularly those that inhibit the expression of ZPI or HCII, are suitable for the treatment, or treatment of, acquired hemophilia. In certain embodiments, the nucleic acids of the invention, particularly those that inhibit the expression of ZPI or HCII, are suitable for the treatment, or treatment of, von Willebrand disease. In certain embodiments, the nucleic acids of the invention, particularly those that inhibit the expression of ZPI or HCII, are suitable for the treatment, or treatment of, parahemophilia.

[0320] Although not wishing to be bound by theory, treatment with the nucleic acids of the invention can result in an increase in coagulation factor levels such that bleeding can be reduced or prevented. Thus, in a preferred embodiment, treatment with a nucleic acid of the invention, particularly a nucleic acid that inhibits the expression of ZPI or HCII, can reduce or prevent bleeding episodes in a subject suffering from hemophilia. In another preferred embodiment, treatment with a nucleic acid of the invention, particularly a nucleic acid that inhibits the expression of ZPI or HCII, can reduce or prevent bleeding into joints of a subject suffering from hemophilia. In certain embodiments, treatment with a nucleic acid of the invention, particularly a nucleic acid that inhibits the expression of ZPI or HCII, can reduce or prevent bleeding into muscle or the brain of a subject suffering from hemophilia.

[0321] The disease to be treated can be diabetes, particularly when the target gene is B4GALT1 as disclosed herein.

[0322] According to the present invention, the term "diabetes" as used herein refers to a group of metabolic diseases in which a subject has hyperglycemia because the body does not produce sufficient insulin or the cells do not respond to the insulin produced. There are three main types of diabetes: (1) Type 1 diabetes (T1D): caused by the body's inability to produce insulin, and the person currently needs to inject insulin (also called insulin-dependent diabetes mellitus, abbreviated IDDM, and juvenile diabetes). (2) Type 2 diabetes (T2D): results from insulin resistance, a condition in which cells cannot properly use insulin, and is sometimes combined with absolute insulin deficiency (formerly called non-insulin-dependent diabetes mellitus, abbreviated NIDDM, and adult-onset diabetes). (3) Gestational diabetes (GD): a condition in which a pregnant woman who has never had diabetes before has high blood glucose levels during pregnancy. This can precede the onset of T2D.

[0323] In certain embodiments, a nucleic acid according to the invention, particularly a nucleic acid that inhibits the expression of B4GALT1, or a pharmaceutical composition comprising said nucleic acid, is used for the treatment of diabetes, preferably type 2 diabetes (T2D).

[0324] The disease to be treated can be a cardiovascular disease, especially when the target gene is B4GALT1 as disclosed herein.

[0325] As used herein, the term "cardiovascular disease" refers to any condition, disorder or disease state associated with, resulting from or causing a structural or functional abnormality that impairs the normal function of the heart or the blood vessels supplying the heart. Cardiovascular diseases can include coronary artery disease, atherosclerosis, myocardial infarction, arteriosclerosis, hypertension, angina, deep vein thrombosis, stroke, congestive heart failure or arrhythmia. In a preferred embodiment, the cardiovascular disease is coronary artery disease.

[0326] In certain embodiments, the nucleic acids according to the invention, in particular nucleic acids that inhibit the expression of B4GALT1, or pharmaceutical compositions comprising said nucleic acids, are used for the treatment of cardiovascular diseases, preferably coronary artery disease.

[0327] The nucleic acids of the invention, such as siRNA, may be administered as "free" nucleic acids 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, prolamine, 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.

[0328] Alternatively, the nucleic acids of the invention, such as siRNA, may be administered as a pharmaceutical composition, such as a dsiRNA liposome formulation.

[0329] In one embodiment, the method includes administering a composition characterized herein such that the expression of the target gene is reduced, for example, by about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the expression of the target gene is reduced for a long period of time, for example, at least about 2, 3, 4 days or more, for example, about 1 week, 2 weeks, 3 weeks, or 4 weeks or longer, for example, about 1 month, 2 months, or 3 months.

[0330] For a subject, a therapeutic amount of a nucleic acid, such as siRNA, can be administered, such as from about 0.01 mg / kg to about 200 mg / kg, to treat a disease related to a hemostatic disorder, such as a disease related to a hemostatic disorder such as hemophilia, or to prevent or treat diabetes or cardiovascular disease.

[0331] The nucleic acid, such as siRNA, can be administered by intravenous infusion periodically over a period of time. In certain embodiments, after an initial treatment regimen, the treatment can be administered at a less frequent rate. Administration of siRNA can reduce the gene product level of the target gene in, for example, the cells or tissues of the patient to at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the detection level of the assay method used. In certain embodiments, the administration results in clinical stabilization or preferably a clinically relevant decrease in at least one sign or symptom of the target gene-related disorder.

[0332] Alternatively, a nucleic acid, such as siRNA, can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver the desired daily dose of nucleic acid, such as siRNA, to a subject. The injections may be repeated over a period of time. The administration may be repeated periodically. In certain embodiments, after an initial treatment regimen, the treatment can be administered at a less frequent rate. A repeated dosing regimen can include the administration of a therapeutic amount of nucleic acid, such as at regular intervals such as every other day or once a year. In certain embodiments, the nucleic acid is administered from about once a month to about once every three months (i.e., about once every quarter).

[0333] In one aspect, the present invention can be applied in compounds, methods, compositions or uses of the following sentences numbered from 1 to 101, and any reference to an equation in Sentences 1 to 101 refers only to the equations defined within Sentences 1 to 101. These equations are reproduced in Figure 5. Specifically, the oligonucleoside moiety represented by Z in any of the following sentences may include a nucleic acid for inhibiting the expression of ZPI, HCII or B4GALT1 as defined below in this specification.

[0334] 1. The following structure:

[0335] [Chemical formula]

[0336] [wherein, each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl and ethyl, R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo and nitro, each occurrence of X1 and X2 is 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 (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 moiety] A compound comprising.

[0337] 2. The compound according to the first sentence, wherein each occurrence of R1 is hydrogen.

[0338] 3. The compound according to the first sentence, wherein R1 is methyl.

[0339] 4. The compound according to the first sentence, wherein R1 is ethyl.

[0340] 5. The compound according to any one of the first to fourth sentences, wherein R2 is hydroxy.

[0341] 6. The compound according to any one of the first to fourth sentences, wherein R2 is halo.

[0342] 7. The compound according to the sixth sentence, wherein R2 is fluoro.

[0343] 8. The compound according to the sixth sentence, wherein R2 is chloro.

[0344] 9. The compound according to the sixth sentence, wherein R2 is bromo.

[0345] 10. The compound according to the sixth sentence, wherein R2 is iodo.

[0346] 11. The compound according to the sixth sentence, wherein R2 is nitro.

[0347] 12. The compound according to any one of the first to eleventh sentences, wherein X1 is methylene.

[0348] 13. The compound according to any one of the first to eleventh sentences, wherein X1 is oxygen.

[0349] 2. A compound according to any one of the first to eleventh sentences, wherein X1 is sulfur.

[0350] 15. A compound according to any one of the first to fourteenth sentences, wherein X2 is methylene.

[0351] 16. A compound according to any one of the first to fifteenth sentences, wherein X2 is oxygen.

[0352] 17. A compound according to any one of the first to sixteenth sentences, wherein X2 is sulfur.

[0353] 18. A compound according to any one of the first to seventeenth sentences, wherein m = 3.

[0354] 19. A compound according to any one of the first to eighteenth sentences, wherein n = 6.

[0355] 20. A compound according to the thirteenth and fifteenth sentences, wherein X1 is oxygen, X2 is methylene, and preferably, q = 1, r = 2, s = 1, t = 1, v = 1. A compound according to the thirteenth and fifteenth sentences.

[0356] 21. A compound according to the twelfth and fifteenth sentences, wherein both X1 and X2 are methylene, and preferably, q = 1, r = 3, s = 1, t = 1, v = 1. A compound according to the twelfth and fifteenth sentences.

[0357] 22. Z is,

[0358]

Chemical formula

[0359] [wherein, Z1, Z2, Z3, and Z4 are each, independently, oxygen or sulfur each time they appear, One of the bond between P and Z2 and the bond between P and Z3 is a single bond, and the other bond is a double bond] The compound according to any one of the first to twenty-first sentences.

[0360] 23. The compound according to sentence 22, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0361] 24. The compound according to 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 the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, and each of the first and second strands having 5' and 3' ends.

[0362] 25. The compound according to sentence 24, wherein the RNA compound has a phosphate attached to the 5' end of its second strand adjacent to the phosphate.

[0363] 26. The compound according to sentence 24, wherein the RNA compound has a phosphate attached to the 3' end of its second strand adjacent to the phosphate.

[0364] 27. The compound of formula (II):

[0365]

Chemical formula

[0366] 28. The compound of formula (III):

[0367]

Chemical formula

[0368] 29. The compound according to claim 27 or 28, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand.

[0369] 30. A composition comprising a compound of formula (II) as defined in claim 27 and a compound of formula (III) as defined in claim 28, optionally dependent on claim 29.

[0370] 31. The composition according to claim 30, wherein the compound of formula (III) as defined in claim 28 is present in an amount in the range of 10 to 15% by weight of the composition.

[0371] 32. A compound of formula (IV):

[0372]

Chemical formula

[0373] 33. A compound of formula (V):

[0374]

Chemical formula

[0375] 34. The compound according to claim 32 or 33, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand.

[0376] 35. A composition comprising a compound of formula (IV) as defined in claim 32 and a compound of formula (V) as defined in claim 33, optionally dependent on claim 34.

[0377] 36. The composition according to claim 35, wherein the compound of formula (V) as defined in claim 33 is present in an amount in the range of 10 to 15% by weight of the composition.

[0378] 37. A compound as defined in any one of claims 1 to 29, or claims 32 to 34, comprising an RNA duplex further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0379] 38. The compound according to claim 37, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0380] 39. A compound as defined in any one of claims 1 to 29, or claims 32 to 34, or claims 37 to 38, wherein the oligonucleoside further comprises one or more protecting moieties at one or more termini.

[0381] 40. The compound according to claim 39, wherein the one or more protecting moieties are not present at the termini of the oligonucleoside chain carrying the ligand moiety, and / or the one or more 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 terminus of the chain carrying the ligand moiety.

[0382] 41. The compound according to any one of claims 1 to 29, or claims 32 to 34, or claims 37 to 40, wherein the ligand moiety shown in formula (I) in claim 1 comprises one or more ligands.

[0383] 42. The compound according to claim 41, wherein the ligand moiety shown in formula (I) in claim 1 comprises one or more carbohydrate ligands.

[0384] 43. The compound according to sentence 42, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides or polysaccharides.

[0385] 44. The compound according to 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.

[0386] 45. The compound according to sentence 44, wherein the one or more carbohydrates comprise one or more N-acetyl-galactosamine moieties.

[0387] 46. The compound according to sentence 45, comprising two or three N-acetylgalactosamine moieties.

[0388] 47. The compound according to any one of sentences 41 to 46, wherein the one or more ligands are attached in a linear configuration or a branched configuration.

[0389] 48. The compound according to sentence 47, wherein the one or more ligands are attached as a bifurcated or trifurcated branched configuration.

[0390] 49. The moiety shown in formula (I) in sentence 1:

[0391]

Chemical formula

[0392] is any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa):

[0393]

Chemical formula

[0394] [wherein, A Iis hydrogen or a suitable hydroxy protecting group, a is an integer of 2 or 3, b is an integer of 2 to 5, or

[0395] [Chemical formula]

[0396] [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 of 1 to 6, or

[0397] [Chemical formula]

[0398] [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 of 2 to 10] is the compound described in Sentences 46 to 48.

[0399] 50. The said moiety shown in formula (I) in Sentence 1:

[0400] [Chemical formula]

[0401] is formula (VII):

[0402] [Chemical formula]

[0403] [In the formula, A I is hydrogen, a is an integer of 2 or 3 is the compound according to the 46th to 48th sentences.

[0404] 51. The compound according to the 49th or 50th sentence, where a = 2.

[0405] 52. The compound according to the 49th or 50th sentence, where a = 3.

[0406] 53. The compound according to the 49th sentence, where b = 3.

[0407] 54. The compound of formula (VIII):

[0408]

Chemical formula

[0409] 55. The compound of formula (IX):

[0410]

Chemical formula

[0411] 56. The compound according to the 54th or 55th sentence, wherein the oligonucleoside comprises an RNA duplex containing a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand.

[0412] 57. A composition comprising the compound of formula (VIII) defined in the 54th sentence and the compound of formula (IX) defined in the 55th sentence, optionally dependent on the 56th sentence.

[0413] 58. The composition according to the 57th sentence, wherein the compound of formula (IX) defined in the 55th sentence is present in an amount in the range of 10 to 15% by weight of the composition.

[0414] 59. Compound of formula (X):

[0415] [Chemical formula]

[0416] 60. Compound of formula (XI):

[0417] [Chemical formula]

[0418] 61. The compound according to paragraph 59 or 60, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand.

[0419]

[0420] 63. The composition according to paragraph 62, wherein the compound of formula (XI) defined in paragraph 60 is present in an amount in the range of 10 to 15% by weight of the composition.

[0421] 64. The compound according to any one of paragraphs 54 to 63, wherein the oligonucleoside further comprises an RNA duplex comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0422] 65. The compound according to paragraph 64, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0423] 66. A compound according to any one of paragraphs 54 to 65, wherein the oligonucleoside further comprises one or more deprotecting moieties at one or more termini.

[0424] 67. The one or more deprotecting moieties are not present at the termini of the oligonucleoside chain carrying the ligand moiety and / or the one or more deprotecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages and inverted abasic nucleosides, and the inverted abasic nucleoside is present at the distal terminus of the chain carrying the ligand moiety as shown in any one of formula (VIII), (IX), (X) or (XI) in any one of paragraphs 54, 55, 59 or 60, a compound according to paragraph 66.

[0425] 68. Compounds of formula (XII) and (XIII):

[0426]

Chemical formula

[0427] [wherein, each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl and ethyl, R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo and nitro, each occurrence of X1 and X2 is 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, v are independently integers from 0 to 4, provided that (i) q and r cannot both be 0 simultaneously, and (ii) s, t and v cannot all be 0 simultaneously, Z is an oligonucleoside moiety] A method for preparing the compound according to any one of the first to 29th, 32nd to 34th, 37th to 56th, 59th to 61st, and 64th to 67th sentences, and / or the composition according to any one of the 30th, 31st, 35th, 36th, 57th, 58th, 62nd, and 63rd sentences, comprising the step of reacting, and, where appropriate, the step of deprotecting the ligand and / or annealing the second strand for the oligonucleoside moiety.

[0428] 69. The compound of formula (XII) is the compounds of formulas (XIV) and (XV):

[0429]

Chemical formula

[0430] [wherein, Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl, R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo, and nitro, Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen, and sulfur, q, r, s, t, v are independently integers from 0 to 4, provided that (i) q and r cannot both be 0 simultaneously, and (ii) s, t, and v cannot all be 0 simultaneously, Z is an oligonucleoside moiety] The method according to sentence 68, which is prepared by reacting.

[0431] 70. The compound of formula (XII) is of formula (XIIa):

[0432]

Chemical formula

[0433] wherein the compound of formula (XIII) is of formula (XIIIa):

[0434]

Chem.

[0435] wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' ends, and the RNA duplex is attached at the 5' end of its second strand to an adjacent phosphate, a compound according to any one of sentences 20, 25, 27, 29, 54, 56, and / or a composition according to any one of sentences 30, 31, 57, 58, and a method according to sentence 68 for preparing the same.

[0436] 71. The compound of formula (XII) is of formula (XIIb):

[0437]

Chem.

[0438] wherein the compound of formula (XIII) is of formula (XIIIa):

[0439]

Chem.

[0440] and the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having 5' and 3' termini, and the RNA duplex being attached at the 5' terminus of its second strand to an adjacent phosphate, a compound according to any one of the 20th, 25th, 28th, 29th, 55th, 56th sentences, and / or a composition according to any one of the 30th, 31st, 57th, 58th sentences, a method according to the 68th sentence for preparing the same.

[0441] 72. The compound of formula (XII) is of formula (XIIc):

[0442]

Chemical formula

[0443] and the compound of formula (XIII) is of formula (XIIIa):

[0444]

Chemical formula

[0445] and the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having 5' and 3' termini, and the RNA duplex being attached at the 3' terminus of its second strand to an adjacent phosphate, a compound according to any one of the 21st, 26th, 32nd, 34th, 59th, 61st sentences, and / or a composition according to any one of the 35th, 36th, 62nd, 63rd sentences, a method according to the 68th sentence for preparing the same.

[0446] 73. The compound of formula (XII) is of formula (XIId):

[0447] [Chemical formula]

[0448] and the compound of formula (XIII) is of formula (XIIIa):

[0449] [Chemical formula]

[0450] wherein the oligonucleoside comprises an RNA double strand containing a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' ends, and the RNA double strand is attached to the adjacent phosphate at the 3' end of its second strand, a compound according to any one of sentences 21, 26, 33, 34, 60, 61, and / or a composition according to any one of sentences 35, 36, 62, 63, the method according to sentence 68 for preparing the same.

[0451] 74. The compound of formula (XIIIa) is of formula (XIIIb):

[0452] [Chemical formula]

[0453] the method according to any one of sentences 70 to 73.

[0454] 75. The compound of formula (XIV) is either of formula (XIVa) or formula (XIVb):

[0455] [Chemical formula]

[0456] and the compound of formula (XV) is either of formula (XVa) or formula (XIVb):

[0457] [Chem.]

[0458] which is such that the oligonucleoside comprises an RNA duplex containing a first and a second strand, the first strand being at least partially complementary to the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having 5' and 3' termini, and (i) the RNA duplex is attached at the 5' terminus of its second strand to an adjacent phosphate in formula (XVa), or (ii) the RNA duplex is attached at the 3' terminus of its second strand to an adjacent phosphate in formula (XVb), the method according to claim 69, dependent on claims 70 to 73.

[0459] 76. A compound of formula (XII):

[0460] [Chem.]

[0461] [wherein R1 is independently selected each time it occurs from the group consisting of hydrogen, methyl and ethyl, R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo and nitro, X1 and X2 are independently selected each time they occur from the group consisting of methylene, oxygen and sulfur, q, r, s, t, v are independently integers from 0 to 4, provided that (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 moiety].

[0462] 77. A compound of formula (XIIa):

[0463]

Chem.

[0464] 78. Compound of formula (XIIb):

[0465]

Chem.

[0466] 79. Compound of formula (XIIc):

[0467]

Chem.

[0468] 80. Compound of formula (XIId):

[0469]

Chem.

[0470] 81. Compound of formula (XIII):

[0471]

Chem.

[0472] [wherein, R1 is independently selected from the group consisting of hydrogen, methyl and ethyl each time it appears, m is an integer from 1 to 6, n is an integer from 1 to 10].

[0473] 82. Compound of formula (XIIIa):

[0474]

Chem.

[0475] 83. Compound of formula (XIIIb):

[0476]

Chem.

[0477] 84. Compound of formula (XIV):

[0478]

Chem.

[0479] [Wherein, R1 is selected from the group consisting of hydrogen, methyl and ethyl, R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo and nitro, X2 is selected from the group consisting of methylene, oxygen and sulfur, s, t, v are independently integers from 0 to 4, provided that s, t and v cannot all be 0 simultaneously].

[0480] 85. Compound of formula (XIVa):

[0481]

Chem.

[0482] 86. Compound of formula (XIVb):

[0483]

Chem.

[0484] 87. Compound of formula (XV):

[0485]

Chem.

[0486] [wherein, each occurrence of R1 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 integers from 0 to 4, provided that q and r cannot both be 0 at the same time, and Z is an oligonucleoside moiety].

[0487] 88. Compound of formula (XV a):

[0488] [Chemical formula]

[0489] 89. Compound of formula (XV b):

[0490] [Chemical formula]

[0491] 90. Use of a compound described in any one of Sentences 1 to 29, Sentences 32 to 34, Sentences 37 to 56, Sentences 59 to 61, and Sentences 64 to 67, and / or a composition described in any one of Sentences 30, 31, 35, 36, 57, 58, 62, and 63, for preparing a compound described in any one of Sentences 76, 81 to 84, and 87.

[0492] 91. Use of a compound described in any one of Sentences 1 to 29, Sentences 32 to 34, Sentences 37 to 56, Sentences 59 to 61, and Sentences 64 to 67, and / or a composition described in any one of Sentences 30, 31, 35, 36, 57, 58, 62, and 63, wherein R2 = F, for preparing a compound described in Sentence 85.

[0493] Use of the compound according to any one of the first to twenty-ninth, thirty-second to thirty-fourth, thirty-seventh to fifty-sixth, fifty-ninth to sixty-first, and sixty-fourth to sixty-seventh sentences, wherein R2 = OH, and / or the composition according to any one of the thirtieth, thirty-first, thirty-fifth, thirty-sixth, fifty-seventh, fifty-eighth, sixty-second, and sixty-third sentences for preparing the compound according to the eighty-sixth sentence.

[0494] 93. Use of the compound according to any one of the twentieth, twenty-fifth, twenty-seventh, twenty-ninth, fifty-fourth, and fifty-sixth sentences, and / or the composition according to any one of the thirtieth, thirty-first, fifty-seventh, and fifty-eighth sentences for preparing the compound according to the seventy-seventh sentence.

[0495] 94. Use of the compound according to any one of the twentieth, twenty-fifth, twenty-eighth, twenty-ninth, fifty-fifth, and fifty-sixth sentences, and / or the composition according to any one of the thirtieth, thirty-first, fifty-seventh, and fifty-eighth sentences for preparing the compound according to the seventy-eighth sentence.

[0496] 95. Use of the compound according to any one of the twenty-first, twenty-sixth, thirty-second, thirty-fourth, fifty-ninth, and sixty-first sentences, and / or the composition according to any one of the thirty-fifth, thirty-sixth, sixty-second, and sixty-third sentences for preparing the compound according to the seventy-ninth sentence.

[0497] 96. Use of the compound according to any one of the twenty-first, twenty-sixth, thirty-third, thirty-fourth, sixtieth, and sixty-first sentences, and / or the composition according to any one of the thirty-fifth, thirty-sixth, sixty-second, and sixty-third sentences for preparing the compound according to the eightieth sentence.

[0498] 97. Use of the compound according to any one of the twentieth, twenty-fifth, twenty-seventh to twenty-ninth, fifty-fourth to fifty-sixth sentences, and / or the composition according to any one of the thirtieth, thirty-first, fifty-seventh, and fifty-eighth sentences for preparing the compound according to the eighty-eighth sentence.

[0499] 98. Use of the compound described in any one of Article 21, Article 26, Article 32 to Article 34, Article 59 to Article 61, and / or the composition described in any one of Article 35, Article 36, Article 62, Article 63 for preparing the compound described in Article 89.

[0500] 99. A compound or composition obtained by or obtainable by the method described in any one of Article 68 to Article 75.

[0501] 100. A pharmaceutical composition comprising the compound described in any one of Article 1 to Article 29, Article 32 to Article 34, Article 37 to Article 56, Article 59 to Article 61, and Article 64 to Article 67, and / or the composition described in any one of Article 30, Article 31, Article 35, Article 36, Article 57, Article 58, Article 62 and Article 63, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0502] 101. The compound described in any one of Article 1 to Article 29, Article 32 to Article 34, Article 37 to Article 56, Article 59 to Article 61, and Article 64 to Article 67, and / or the composition described in any one of Article 30, Article 31, Article 35, Article 36, Article 57, Article 58, Article 62 and Article 63 for use in therapy.

[0503] In another aspect, the present invention can be applied to the compounds, methods, compositions or uses of the following items numbered 1 to 56, and any reference to an arbitrary formula in the items refers only to the formula defined within Items 1 to 56. These formulas are reproduced in FIG. 6. Specifically, the oligonucleoside moiety represented by Z in any of the following items may contain a nucleic acid for inhibiting the expression of ZPI, HCII or B4GALT1 as defined below in the present specification.

[0504] 1. The following structure:

[0505]

Chemical formula

[0506] [wherein r and s are independently integers selected from 1 to 16, Z is an oligonucleoside moiety] A compound comprising

[0507] 2. The compound according to claim 1, wherein s is an integer selected from 4 to 12.

[0508] 3. The compound according to claim 2, wherein s is 6.

[0509] 4. The compound according to any one of claims 1 to 3, wherein r is an integer selected from 4 to 14.

[0510] 5. The compound according to claim 4, wherein r is 6.

[0511] 6. The compound according to claim 4, wherein r is 12.

[0512] 7. The compound according to claim 5, which depends on claim 3.

[0513] 8. The compound according to claim 6, which depends on claim 3.

[0514] 9. Z is

[0515] [Chemical formula]

[0516] [wherein Z1, Z2, Z3, Z4 are independently oxygen or sulfur each time they appear, One of the bond between P and Z2 and the bond between P and Z3 is a single bond, and the other bond is a double bond] The compound according to any one of claims 1 to 8.

[0517] 10. The compound according to any one of claims 1 to 9, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0518] 11. The compound according to claim 10, wherein the RNA compound comprises an RNA duplex containing a first and a second strand, the first strand being at least partially complementary to the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, and each of the first and second strands having 5' and 3' ends.

[0519] 12. The compound according to claim 11, wherein the RNA compound is attached to an adjacent phosphate at the 5' end of its second strand, preferably also being dependent on claims 3 and 6.

[0520] 13. The compound according to claim 11, wherein the RNA compound is attached to an adjacent phosphate at the 3' end of its second strand, preferably also being dependent on claims 3 and 5.

[0521] 14. A compound of formula (II), preferably being dependent on claim 12:

[0522]

Chemical formula

[0523] 15. A compound of formula (III), preferably being dependent on claim 13:

[0524]

Chemical formula

[0525] 16. The compound as defined in any one of claims 1 to 15, wherein the oligonucleoside further comprises one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position, in an RNA duplex.

[0526] 17. The compound according to claim 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0527] 18. The compound according to any one of claims 1 to 17, wherein the oligonucleoside further comprises one or more deprotecting moieties at one or more termini.

[0528] 19. The one or more deprotecting moieties are not present at the termini of the oligonucleoside chain carrying the linker / ligand moiety, and / or the one or more deprotecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleoside is present at the distal terminus of the same chain relative to the terminus carrying the linker / ligand moiety. The compound according to claim 18.

[0529] 20. The compound according to any one of claims 1 to 19, wherein the ligand moiety represented by formula (I) in claim 1 comprises one or more ligands.

[0530] 21. The compound according to claim 20, wherein the ligand moiety represented by formula (I) in claim 1 comprises one or more carbohydrate ligands.

[0531] 22. The compound according to claim 21, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[0532] 23. The compound according to claim 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.

[0533] 24. The compound according to claim 23, wherein the one or more carbohydrates comprise one or more N-acetyl-galactosamine moieties.

[0534] 25. The compound according to claim 24, comprising two or three N-acetylgalactosamine moieties.

[0535] 26. The compound according to any one of the preceding claims, wherein the one or more ligands are attached in a linear configuration or a branched configuration.

[0536] 27. The compound according to claim 26, wherein the one or more ligands are attached as a bifurcated or trifurcated branched configuration.

[0537] 28. The moiety represented by formula (I) in claim 1:

[0538]

Chemical formula

[0539] is any one of formula (IV), (V) or (VI), preferably formula (IV):

[0540]

Chemical formula

[0541] [wherein, 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

[0542]

Chemical formula

[0543] [wherein, 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

[0544] [Chemical formula]

[0545] [wherein, A I is hydrogen or a suitable hydroxy protecting group, a is an integer of 2 or 3, e is an integer of 2 to 10] is the compound according to any one of claims 20 to 27.

[0546] 29. The said moiety represented by formula (I) in claim 1:

[0547] [Chemical formula]

[0548] is formula (VII):

[0549] [Chemical formula]

[0550] [wherein, A I is hydrogen, a is an integer of 2 or 3] is the compound according to any one of claims 1 to 28.

[0551] 30. The compound according to claim 28 or 29, wherein a = 2.

[0552] 31. The compound according to claim 28 or 29, wherein a = 3.

[0553] 32. The compound according to claim 28, wherein b = 3.

[0554] 33. The compound of formula (VIII):

[0555] [Chemical formula]

[0556] 34. Compound of formula (IX):

[0557]

Chemical formula

[0558] 35. The compound according to item 33 or 34, comprising an RNA duplex in which the oligonucleoside further comprises one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0559] 36. The compound according to item 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0560] 37. The compound according to any one of items 33 to 36, wherein the oligonucleoside further comprises one or more deprotection moieties at one or more termini.

[0561] 38. The one or more deprotection moieties are not present at the termini of the oligonucleoside chain carrying the linker / ligand moiety, and / or the one or more deprotection moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleoside is present at the distal terminus of the same chain with respect to the terminus carrying the linker / ligand moiety. The compound according to item 37.

[0562] 39. The oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' termini, and the RNA duplex is attached to an adjacent phosphate at the 5' terminus of its second strand. The compound according to item 33.

[0563] 40. The compound according to item 34, wherein the oligonucleoside comprises an RNA duplex containing a first and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first and second strands has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand.

[0564] 41. Compounds of formula (X) and (XI):

[0565]

Chemical formula

[0566] [Wherein, r and s are independently integers selected from 1 to 16, Z is an oligonucleoside moiety] A method for preparing the compound according to any one of items 1 to 40, comprising the step of reacting, and, if appropriate, deprotecting the ligand and / or annealing the second strand for the oligonucleoside.

[0567] 42. The compound of formula (X) is of formula (Xa):

[0568]

Chemical formula

[0569] and the compound of formula (XI) is of formula (XIa):

[0570]

Chemical formula

[0571] and the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having 5' and 3' termini, and the RNA duplex being attached at the 5' terminus of its second strand to an adjacent phosphate, a method according to claim 41 for preparing a compound according to any one of claims 6, 8 to 14, 16 to 33, and 35 to 40.

[0572] 43. The compound of formula (X) is of formula (Xb):

[0573]

Chemical formula

[0574] and the compound of formula (XI) is of formula (XIa):

[0575]

Chemical formula

[0576] and the oligonucleoside comprises an RNA duplex comprising a first and a second strand, the first strand being at least partially complementary to the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, each of the first and second strands having 5' and 3' termini, and the RNA duplex being attached at the 3' terminus of its second strand to an adjacent phosphate, a method according to claim 41 for preparing a compound according to any one of claims 5, 7, 9 to 13, 15 to 32, and 34 to 40.

[0577] 44. The compound of formula (XIa) is of formula (XIb):

[0578]

Chemical formula

[0579] The method according to claim 42 or 43, which is

[0580] 45. A compound of formula (X):

[0581] [Chemical formula]

[0582] [wherein, r is independently an integer selected from 1 to 16, Z is an oligonucleoside moiety].

[0583] 46. A compound of formula (Xa):

[0584] [Chemical formula]

[0585] 47. A compound of formula (Xb):

[0586] [Chemical formula]

[0587] 48. A compound of formula (XI):

[0588] [Chemical formula]

[0589] [wherein, s is independently an integer selected from 1 to 16, Z is an oligonucleoside moiety].

[0590] 49. A compound of formula (XIa):

[0591] [Chemical formula]

[0592] 50. Compound of formula (XIb):

[0593] [Chemical formula]

[0594] 51. Use of a compound according to any one of claims 45 and 48 to 50 for preparing a compound according to any one of claims 1 to 40.

[0595] 52. Use of a compound according to claim 46 for preparing a compound according to any one of claims 6, 8 to 14, 16 to 33, and 35 to 40.

[0596] 53. Use of a compound according to claim 47 for preparing a compound according to any one of claims 5, 7, 9 to 13, 15 to 32, and 34 to 40.

[0597] 54. A compound or composition obtained or obtainable by a method according to any one of claims 41 to 44.

[0598] 55. A pharmaceutical composition comprising a compound according to any one of claims 1 to 40 together with a pharmaceutically acceptable carrier, diluent or excipient.

[0599] 56. A compound according to any one of claims 1 to 40 for use in therapy. [Examples]

[0600] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes contemplated in light thereof are proposed to those skilled in the art and should be included within the spirit and scope of the present application and the scope of the appended claims.

[0601] Example 1: Synthesis of Tether 1 General experimental conditions: Thin layer chromatography (TLC) was performed on aluminum plates coated with silica using a fluorescence indicator 254 nm from Macherey-Nagel. Compounds were visualized under UV light (254 nm) or after spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent from Stahl (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).

[0602] All moisture-sensitive reactions were carried out under anhydrous conditions using dried glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH+Co. KG. D-Galactosamine pentaacetate was purchased from AK scientific.

[0603] HPLC / ESI-MS was performed at 60 °C using an Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1×100 mm) from Waters on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A with H2O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5 - 100% B over 15 minutes at a flow rate of 0.4 mL / min was used. Detector and conditions: Corona charged aerosol detection (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0604] 1 H and 13 C NMR spectra were recorded 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, referenced to the solvent residual peak (CDCl3 - 1 H NMR: δ at 7.26 ppm, and 13 C NMR δ at 77.2 ppm; DMSO-d6 - 1H 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 described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[0605] Synthetic route for the conjugate building block TriGalNAc_tether1:

[0606]

Chem.

[0607] Preparation of Compound 2: Pentaacetyl-D-galactosamine (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 NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0 - 10% MeOH in DCM, 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[0608]

Chem.

[0609] 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 hour. Then, TMSOTf (0.77 g, 3.49 mmol, 0.5 equiv) was added to the mixture, and the reaction was stirred overnight. The molecular sieves were filtered off, 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 for 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 for, 504.21. Found 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).

[0610]

Chem.

[0611] 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 (3×) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through Celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to give the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: C 20 H 34 N2O 11 Calculated for, 478.2. Found 479.4.

[0612]

Chem.

[0613] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 equiv) was dissolved in a mixture of DCM / water (40 mL, 1:1 v / v), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 equiv) was added 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 CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure, and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% EtOAc in cyclohexane with 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).

[0614]

Chem.

[0615] Preparation of Compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 equiv) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to obtain 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

[0616]

Chem.

[0617] Preparation of Compound 9: CbzNH-Tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 equiv) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 equiv) were dissolved in N,N-dimethylformamide (DMF) (25 mL). Then, N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 equiv), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 equiv) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 equiv) were added to the solution and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL) and washed with saturated aqueous NaHCO3 solution (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated and the crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM for 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 calcd for, 1852.9. Found 1854.7. 1 H NMR (500 MHz, DMSO-d6) δ 7.90 - 7.80 (m, 10H), 7.65 - 7.62 (m, 4H), 7.47 - 7.43 (m, 3H), 7.38 - 7.32 (m, 8H), 5.24 - 5.22 (m, 3H), 5.02 - 4.97 (m, 4H), 4.60 - 4.57 (m, 3 H), 4.07 - 3.90 (m 10H), 3.67 - 3.36 (m, 70H), 3.23 - 3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80 - 1.78 (m, 17H). 1313C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[0618]

Chem.

[0619] Preparation of Compound 10: The trifurcated 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 (3×) and hydrogenated overnight under balloon pressure. Mass spectrometry was performed following the completion of the reaction, and the resulting mixture was filtered through a thin pad of celite. The solvent was evaporated, and the obtained residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellowish oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 Calculated value of, 1718.8. Measured value 1719.3.

[0620]

Chem.

[0621] Preparation of Compound 11: Commercially available bis(N-hydroxysuccinimide ester) suberate (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, a solution of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0 equiv) in DMF (5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 3 hours. 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 for 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 for C H N5O5, 353.4. Found 354.3.

[0622]

Chemical Structure

[0623] Preparation of TriGalNAc(12): The 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 mixture was stirred at room temperature overnight. 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 for 20 CV) to obtain 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 for, 1957.1. Found 1959.6.

[0624] Conjugation of Tether 1 to siRNA strands: Monofluorocyclooctyne (MFCO) conjugation at the 5' or 3' end 5'-end MFCO conjugation

[0625] [Chemical formula]

[0626] 3'-end MFCO conjugation

[0627] [Chemical formula]

[0628] 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). To this solution, a 35 mM solution of 1 molar equivalent of MFCO-C6-NHS ester (Berry & Associates, catalog number LK4300) in DMF was added. The reaction was carried out at room temperature. After 1 hour, an additional 1 molar equivalent of MFCO solution was added. The reaction was allowed to proceed for a further 1 hour and monitored by LC / MS. At least 2 molar equivalents of excess MFCO NHS ester reagent relative to the amino-modified oligonucleotide were 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).

[0629] Purification was carried out using a Waters XBridge C18 Prep 19×50 mm column. 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. A UV trace at 280 nm was recorded. A gradient of 0 - 100% B was used within 60 column volumes.

[0630] Fractions containing full-length conjugated oligonucleotides were pooled and precipitated in the freezer using 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 obtain the conjugated oligonucleotides in an isolated yield of 40 - 80%.

[0631] 5'-GalNAc-T1 conjugate

[0632]

Chem.

[0633] 3'-GalNAc-T1 conjugate

[0634]

Chem.

[0635] General procedure for TriGalNAc conjugation: The MFCO-modified single strand was dissolved in water at 2000 OD / mL, and a 1 equivalent solution of compound 12 (10 mM) in DMF was added to this solution. The reaction was carried out at room temperature, and after 3 hours, a 0.7 molar equivalent 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).

[0636] RP-HPLC purification was performed using an XBridge C18 Prep 19×50 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. A UV trace at 280 nm was recorded. A gradient of 0–100% B was used within 60 column volumes.

[0637] Fractions containing the full-length conjugated oligonucleotide were pooled and precipitated in the freezer using 3M NaOAc, pH 5.2 and 85% ethanol, and 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.

[0638] The conjugate was desalted by size-exclusion chromatography using an Akta Pure (GE Healthcare) instrument with Sephadex G25 Fine resin (GE Healthcare) to obtain the conjugated oligonucleotide in an isolated yield of 50–70%.

[0639] The following schemes further illustrate the synthetic route: Scheme 1:

[0640] [Chemical formula]

[0641] Scheme 2:

[0642] [Chemical formula]

[0643] Scheme 3:

[0644] [Chemical formula]

[0645] Scheme 4:

[0646]

Chem.

[0647] Scheme 5:

[0648]

Chem.

[0649] Example 2: Double-Strand Annealing To generate the desired siRNA double strand, two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixture was placed in a water bath at 70 °C for 5 minutes and then cooled to ambient temperature within 2 hours. The double strand was lyophilized for 2 days and stored at -20 °C.

[0650] The double strand was 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. A homogeneous concentration gradient was run for 10 minutes 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).

[0651] Example 3: Synthesis of Tether 2 General Experimental Conditions: Thin-layer chromatography (TLC) was performed on aluminum plates coated with silica using a fluorescent indicator 254 nm from Macherey-Nagel. Compounds were visualized under UV light (254 nm) or after spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent from Stahl (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).

[0652] All moisture-sensitive reactions were carried out under anhydrous conditions using dried glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich and the solvents from Carl Roth GmbH + Co. KG. D-Galactosamine pentaacetate was purchased from AK scientific.

[0653] HPLC / ESI-MS was performed at 60 °C using an Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1×100 mm) from Waters on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A with H2O containing 0.1% formic acid and solvent B with acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5 - 100% B over 15 minutes at a flow rate of 0.4 mL / min was used. Detector and conditions: Corona charged aerosol detection (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0654] 1 H and 13 C NMR spectra were recorded at room temperature on a Varian spectrometer at 500 MHz ( 1 H NMR) and 125 MHz (13 recorded by (13)C NMR. Chemical shifts are given in ppm, referenced to the solvent residual peak (CDCl3 - 1 1H NMR: δ at 7.26 ppm, and 13 13C NMR δ at 77.2 ppm; DMSO-d6 - 1 1H NMR: δ at 2.50 ppm, and 13 13C NMR δ at 39.5 ppm). Coupling constants are given in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m).

[0655] Synthetic route for the conjugate building block TriGalNAc - tether2:

[0656]

Chemical Structure

[0657] Preparation of Compound 2: Penta - O - acetyl - D - galactosamine (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 mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aqueous NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0 - 10% MeOH in DCM, 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[0658]

Chemical Structure

[0659] 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 hour. Then, TMSOTf (0.77 g, 3.49 mmol, 0.5 equiv) was added to the mixture, and the reaction was stirred overnight. The molecular sieves were filtered off, 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 for 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 for, 504.21. Found 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).

[0660] [Chemistry]

[0661] Preparation of Compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq) 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 (3×) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through Celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to give the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: C 20 H 34 N2O 11 Calculated for, 478.2. Found 479.4.

[0662] [Chemistry]

[0663] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq) was dissolved in a mixture of DCM / water (40 mL 1:1 v / v), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 eq) was added with vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure, and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% EtOAc in cyclohexane with 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).

[0664]

Chem.

[0665] Preparation of Compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 equiv) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to obtain 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.

[0666]

Chem.

[0667] Preparation of Compound 9: CbzNH-Tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 equiv) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 equiv) were dissolved in N,N-dimethylformamide (DMF) (25 mL). Then, N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 equiv), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 equiv) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 equiv) were added to the solution and the reaction mixture was stirred for 72 h. The solvent was removed under reduced pressure and the residue was dissolved in DCM (100 mL) and washed with saturated aqueous NaHCO3 solution (100 mL). The organic layer was dried over Na2SO4 and the solvent was evaporated. The crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM for 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, 1852.9. Found 1854.7. 1 H NMR (500 MHz, DMSO-d6) δ 7.90 - 7.80 (m, 10H), 7.65 - 7.62 (m, 4H), 7.47 - 7.43 (m, 3H), 7.38 - 7.32 (m, 8H), 5.24 - 5.22 (m, 3H), 5.02 - 4.97 (m, 4H), 4.60 - 4.57 (m, 3 H), 4.07 - 3.90 (m 10H), 3.67 - 3.36 (m, 70H), 3.23 - 3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80 - 1.78 (m, 17H). 1313C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[0668]

Chem.

[0669] Preparation of Compound 10: The 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 (3×) and hydrogenated overnight under balloon pressure. Following completion of the reaction, mass spectrometry was performed, and the resulting mixture was filtered through a thin pad of celite. The solvent was evaporated, and the obtained residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellowish oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 Calculated value, 1718.8. Measured value 1719.3.

[0670]

Chem.

[0671] Preparation of Compound 14: The 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, a solution of compound 13 (0.14 g, 0.53 mmol, 2.0 equiv) in DCM (5 mL) was added dropwise. The reaction was stirred at room temperature overnight. 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 for 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.

[0672]

Chemical formula

[0673] Preparation of TriGalNAc (15): The branched 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 by using a vacuum / argon cycle (3×) and hydrogenated overnight under balloon pressure. The completion of the reaction was monitored by mass spectrometry, and the resulting mixture was filtered through a thin pad of 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: Calculated for C 81 H 131 N7O 42 is 1874.9. Found 1875.3.

[0674] Conjugation of Tether 2 to siRNA Strand: TriGalNAc Tether 2 (GalNAc-T2) conjugation at the 5'-end or 3'-end 5'-GalNAc-T2 conjugate

[0675]

Chem.

[0676] 3'-GalNAc-T2 conjugate

[0677]

Chem.

[0678] 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) was added N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol). The solution was stirred at room temperature for 18 h and used in the subsequent conjugation reaction without further purification.

[0679] 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). To this solution was added 1 molar equivalent of a solution of tether 2 NHS ester (57 mM) in DMF. The reaction was carried out at room temperature and after 1 h, a further 1 molar equivalent of the NHS ester solution was added. The reaction was allowed to proceed for a further 1 h and the progress of the reaction was monitored by LCMS. At least 2 molar equivalents of excess NHS ester reagent relative to the amino-modified oligonucleoside were necessary 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.

[0680] Purification was carried out using an XBridge C18 Prep 19×50mm column from Waters. Buffer A was 100 mM TEAA pH7, and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60 °C were used. A UV trace at 280 nm was recorded. A gradient of 0–100% B was used within 60 column volumes.

[0681] Fractions containing the full-length conjugated oligonucleosides were pooled and precipitated in the freezer using 3M NaOAc, pH 5.2 and 85% ethanol, and then dissolved in water at 1000 OD / mL. O-acetate was removed using 20% ammonium hydroxide in water until completion (monitored by LC-MS).

[0682] The conjugate was desalted by size exclusion chromatography using an Akta Pure (GE Healthcare) instrument with Sephadex G25 Fine resin (GE Healthcare) to obtain the conjugated oligonucleotides in 60–80% isolated yield.

[0683] The conjugate was characterized by HPLC-MS analysis using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics) using a 2.1×50mm XBridge C18 column (Waters). Buffer A was 16.3 mM triethylamine, 100 mM HFIP in 1% MeOH in H2O, 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 1–100% B was used within 31 minutes.

[0684] The following scheme further shows the synthetic route: Scheme 6:

[0685] [Chemistry]

[0686] Scheme 7:

[0687] [Chemistry]

[0688] Scheme 8:

[0689] [Chemistry]

[0690] Scheme 9:

[0691] [Chemistry]

[0692] Example 4: Double-strand annealing To generate the desired siRNA double strand, two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixture was placed in a water bath at 70 °C for 5 minutes and then cooled to ambient temperature within 2 hours. The double strand was lyophilized for 2 days and stored at -20 °C.

[0693] The double-strand was analyzed by analytical SEC-HPLC using 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. A uniform concentration gradient was run for 10 minutes at a flow rate of 1.5 mL / min at room temperature. UV traces were recorded at 260 and 280 nm. 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).

[0694] Example 5: Alternative synthetic route for conjugate building block TriGalNAc-tether 2:

[0695]

Chemical formula

[0696] Conjugation of tether 2 to siRNA strands: TriGalNAc tether 2 (GalNAc-T2) conjugation at the 5'-end or 3'-end Conjugation conditions

[0697]

Chemical formula

[0698] Pre-activation: To a solution of compound 15 (16 μmol, 4 eq) in DMF (160 μL), TFA-O-PFP (15 μl, 21 eq) was added followed by DIPEA (23 μl, 32 eq) at 25 °C. The tube was shaken at 25 °C for 2 h. The reaction was quenched with H2O (10 μL).

[0699] Coupling: The resulting mixture was diluted with DMF (400 μl), followed by addition of an 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 h 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 h. The mixture was analyzed by LCMS, concentrated, and purified by IP-RP HPLC to yield an oligonucleotide conjugated to Tether 2 GalNAc.

[0700] 5'-GalNAc-T2 conjugate

[0701]

Chemical formula

[0702] 3'-GalNAc-T2 conjugate

[0703]

Chemical formula

[0704] Example 6: Solid-phase synthesis method: Scale ≤ 1 μmol The synthesis of the siRNA sense and antisense strands was performed on a MerMade192X synthesizer using a commercially available solid support made from controlled pore glass with a universal linker (Universal CPG, having a loading of 40 μmol / g; LGC Biosearch or Glen Research).

[0705] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[0706] The 2'-O-methyl phosphoramidites used were as follows: 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, 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.

[0707] The 2'-F phosphoramidites used were as follows: 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-deoxythymidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0708] All phosphoramidites, except 2'-O-methyl-uridine phosphoramidite dissolved in DMF / MeCN (1:4, v / v), were dissolved at a concentration of 0.05 M in anhydrous acetonitrile (Honeywell Research Chemicals). 0.02 M iodine (DNAchem) in acetonitrile / pyridine / H2O was used as the oxidizing reagent. Thiolation for phosphorothioate linkage was carried out using 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v. 5-Ethylthiotetrazole (ETT), 0.25 M mM in acetonitrile, was used as the activating solution.

[0709] 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).

[0710] In each cycle, DMT was removed by 3% TCA (DNAchem) in DCM, the deblocking solution.

[0711] The coupling time was 180 seconds. The oxidant contact time was set at 80 seconds and the thiolation time was 2 * 100 seconds.

[0712] At the end of the synthesis, the oligonucleotide was cleaved from the solid support using an NH4OH:EtOH solution 4:1 (v / v) for 20 hours at 45 °C (TCI). The solid support was then filtered off, the filter was washed thoroughly with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[0713] The oligonucleotide was treated using an Amicon Ultra-2 centrifugal filter unit; ultracentrifugation using PBS buffer (10×, Teknova, pH 7.4, sterile), or EtOH precipitation from 1 M sodium acetate, to form the sodium salt.

[0714] The single strand was evaluated by MS ESI−, then annealed in water to form the final double-stranded siRNA, and the purity of the duplex was evaluated by size exclusion chromatography.

[0715] Example 7: Solid-phase synthesis method: scale ≥ 5 μmol The synthesis of the siRNA sense and antisense strands was carried out on a commercially available solid support (Universal CPG, having a loading of 40 μmol / g; LGC Biosearch or Glen Research) made from controlled pore glass using a Universal linker at a scale of 5 μmol on a MerMade12 synthesizer. The sense strand intended for 3'-conjugation was synthesized at 12 μmol on a 3'-PT-amino-modifier C6 CPG 500 Å solid support (LGC) having a loading of 86 μmol / g.

[0716] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[0717] The 2'-O-methyl phosphoramidites used were as follows: 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, 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.

[0718] The 2'-F phosphoramidites used were as follows: 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-deoxythymidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0719] 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).

[0720] All phosphoramidites except 2'-O-methyl-uridine phosphoramidite dissolved in DMF / MeCN (1:4, v / v) were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M. Iodine (DNAchem) at 0.02 M in acetonitrile / pyridine / H2O was used as the oxidizing reagent. Thiolation for phosphorothioate linkage was carried out using 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v. 5-Ethylthiotetrazole (ETT) at 0.25 M mM in acetonitrile was used as the activation solution.

[0721] In each cycle, DMT was removed by 3% TCA (DNAchem) in DCM, the deblocking solution.

[0722] For the strand synthesized on universal CPG, coupling was carried out for 130 seconds using 8 equivalents of amidite. The oxidation time was 47 seconds and the thiolation time was 210 seconds.

[0723] For the strand 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.

[0724] At the end of the synthesis, the oligonucleotide was cleaved from the solid support using an NH4OH:EtOH solution 4:1 (v / v) for 20 hours at 45 °C (TCI). The solid support was then filtered off, the filter was washed thoroughly with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[0725] The oligonucleotide was treated by ethanol precipitation from 1M sodium acetate to form the sodium salt.

[0726] The single-stranded oligonucleotide was purified by IP-RP HPLC on a Xbridge BEH C18 5μm, 130Å, 19×150mm (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.

[0727] The purity and identity of the single-stranded were evaluated by UPLC / MS ESI- on a Xbridge BEH C18 2.5μm, 3×50mm (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).

[0728] The sense strand was conjugated according to the protocol provided in any of Examples 1, 3 or 5.

[0729] Next, the sense and antisense strands were annealed in water to form the final double-stranded siRNA, and the purity of the double-stranded product was evaluated by size-exclusion chromatography.

[0730] Example 8: Nucleic Acid Sequences: The siRNA oligonucleosides suitable for use according to the present invention may target HCII, ZPI or B4GALT1. The complete DNA sequences of the HCII, ZPI and B4GALT1 targets are as follows (SEQ ID NOs: 1, 2 and 3):

[0731] SEQ ID NO: 1 (HCII)

[0732] JPEG2025524135000126.jpg85167JPEG2025524135000127.jpg252167JPEG2025524135000128.jpg252166JPEG2025524135000129.jpg252167JPEG2025524135000130.jpg253167JPEG2025524135000131.jpg139167

[0733] SEQ ID NO: 2 (ZPI)

[0734] JPEG2025524135000132.jpg94166JPEG2025524135000133.jpg253166JPEG2025524135000134.jpg253167JPEG2025524135000135.jpg253167JPEG2025524135000136.jpg253166JPEG2025524135000137.jpg82166

[0735] SEQ ID NO: 3 (B4GALT1)

[0736] JPEG2025524135000138.jpg152168JPEG2025524135000139.jpg252168JPEG2025524135000140.jpg253166JPEG2025524135000141.jpg252167JPEG2025524135000142.jpg253167JPEG2025524135000143.jpg252167JPEG2025524135000144.jpg253168JPEG2025524135000145.jpg253167JPEG2025524135000146.jpg253169JPEG2025524135000147.jpg253167JPEG2025524135000148.jpg253168JPEG2025524135000149.jpg253167JPEG2025524135000150.jpg253168JPEG2025524135000151.jpg253168JPEG2025524135000152.jpg251168JPEG2025524135000153.jpg106167

[0737] Table 1 below provides the oligonucleoside mRNA target sequences of HCII, ZPI, and B4GALT1, along with their corresponding positions in transcripts NM_000185.4 (HCII), NM_016186.3 (ZPI), and NM_001497.4 (B4GALT1).

[0738] [Table 1]

[0739] Table 2 provides, as follows, the sequences of the unmodified first (antisense) strand and the corresponding unmodified second (sense) strand of the siRNA oligonucleotides (targeting HCII, ZPI, and B4GALT1) according to the present invention, along with their corresponding positions in the entire gene sequences of SEQ ID NO: 1, 2, or 3.

[0740] [Table 2]

[0741] Table 3 provides the modified first (antisense) sequences for siRNA oligonucleosides (targeting HCII, ZPI, and B4GALT1) according to the present invention, together with the corresponding unmodified first (antisense) sequences, as follows.

[0742]

Table 3

[0743] Table 4 provides the modified second (sense) sequences for siRNA oligonucleosides (targeting HCII, ZPI, and B4GALT1) according to the present invention, together with the corresponding unmodified second (sense) sequences, as follows.

[0744]

Table 4

[0745] A portion of the sequence of the modified second strand exemplified above in Table 4 includes the preferred 5'iaia motif. However, it should also be understood that the scope of these modified second strand sequences further includes the Me / F modified second strand in the absence of the 5'iaia motif.

[0746] Table 5 identifies duplexes using duplex IDs with reference to the modified antisense and sense IDs from the previous Tables 3 and 4.

[0747]

Table 5

[0748] 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 o - thermolabile modification

[0749] Example 9: Inhibition Screening 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% CO2 atmosphere. The cells are transfected with siRNA duplexes targeting the target gene mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 85), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 84)) 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 purification is performed using the RNeasy 96 Kit (Qiagen). Each duplex is tested by transfection in duplicate wells in two independent experiments.

[0750] cDNA synthesis is performed using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) is 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.

[0751] qPCR is performed in duplicate for the cDNA from each well, and the average Ct is calculated. Relative HCII expression is normalized to GAPDH from the average Ct values using the comparative Ct (ΔΔCt) method and calculated relative to untreated cells. Based on the results of the primary screening, siRNA duplexes showing good activity are selected for dose-response follow-up.

[0752] 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% CO2 atmosphere. Cells are transfected with siRNA duplexes targeting the target gene mRNA or negative control siRNA (siRNA control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 85), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 84)) using 10×3-fold serial dilutions over a final double-stranded concentration range of 20 nM to 1 pM. 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. Cells are incubated at 37°C / 5% CO2 for 24 hours and then total RNA is purified using the RNeasy 96 Kit (Qiagen). Each duplex is tested by transfection in duplicate wells in a single experiment.

[0753] cDNA synthesis is performed using the FastQuant RT (with gDNase) Kit (Tiangen). Real-time quantitative PCR (qPCR) is 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.

[0754] qPCR was performed in duplicate on the cDNA from each well to calculate the average Ct. Relative HCII expression was normalized to GAPDH from the average Ct values using the comparative Ct (ΔΔCt) method and calculated relative to untreated cells. The maximum percent inhibition and IC50 values of HCII expression were calculated using a four-parameter (variable slope) model with GraphPad Prism 9.

[0755] Example 11: 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 siRNA duplexes designed against the target or negative control siRNA at 0.1 nM and 1 nM. Transfection was carried out 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 was incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. 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 3 times.

[0756] 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 an ABI QuantStudio 7.

[0757] qPCR was performed in duplicate for cDNA from each well, and the average Ct was calculated. Relative target expression was normalized to GAPDH from the average Ct values using the comparative Ct (ΔΔCt) method and calculated relative to untreated cells.

[0758] For the inhibition of ZPI, siRNA duplexes ETXM1201 and ETXM1227 were tested (Figure 8). For the inhibition of B4GALT1, siRNA duplexes ETXM1764 and ETXM1231 (Figure 9) and ETXM1772 and ETXM1232 (Figure 10) were tested.

[0759] The present invention is not intended to be limited in scope to the specific disclosed embodiments provided, for example, to illustrate various aspects of the present invention. Various modifications to the described compositions and methods will be apparent from the description and teachings herein. Such variations can be made without departing from the true scope and spirit of the present disclosure and are intended to be within the scope of the present disclosure.

[0760] In the event of any ambiguity between the sequences herein and those in the accompanying sequence listing, the sequences provided herein are considered to be the correct sequences.

Claims

1. A nucleic acid for inhibiting the 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 double-stranded region having a length of at least 17 nucleosides, the second strand contains two consecutive abasic nucleosides in the 5'-terminal region of the second strand, which includes 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, (a) the penultimate abasic nucleoside is connected to the adjacent first base nucleoside in the adjacent 5'-terminal proximal region through a reverse nucleoside internucleoside linkage, (b) the reverse linkage is a 5-5' reverse linkage, and (c) the linkage between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5' when read towards the end containing the terminal abasic nucleoside and the penultimate abasic nucleoside, the first strand contains at least one double-stranded thermally destabilizing modification within the first 9 nucleoside positions of its 5'-region, a nucleic acid.

2. A nucleic acid for inhibiting the 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 double-stranded region having a length of at least 17 nucleosides, and the nucleosides of the second strand are as follows (5'-3'): ia-ia-(Me) 8 -(F) 3 -(Me) 10 [wherein, ia represents a reversed abasic nucleoside] including a 2'-sugar and abasic modification pattern, and the first strand contains at least one double-stranded thermally destabilizing modification within the first 9 nucleoside positions of its 5'-region, a nucleic acid.

3. The nucleic acid according to claim 1 or 2, wherein the destabilizing modification is selected from unlocked nucleic acid (UNA) and glycol nucleic acid (GNA).

4. The nucleic acid according to claim 3, wherein the destabilizing modification contains at least one unlocked nucleic acid (UNA).

5. The nucleic acid according to claim 3, wherein the destabilizing modification contains at least one glycol nucleic acid (GNA), particularly at least one (S)-glycol nucleic acid.

6. The nucleic acid according to any one of claims 1 to 5, wherein the at least one double-strand heat destabilizing modification is located at positions 2 to 9, preferably positions 2 to 8, more preferably positions 3 to 8, still more preferably positions 4 to 8, and most preferably positions 6 or 7 of the nucleosides in the 5'-region of the first strand.

7. The nucleic acid according to any one of claims 1 to 6, wherein the nucleosides of the first strand contain a 2'-sugar modification pattern, and 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 strand is not composed of 4 or 6 2'-F modifications.

8. The nucleic acid according to claim 7, wherein the nucleosides of the first strand contain a 2'-sugar modification pattern, and 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 strand consists of 3, 5 or 7 2'-F modifications.

9. The nucleic acid according to claim 8, wherein the nucleosides of the first strand contain a 2'-sugar modification pattern, and 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 strand consists of 3 2'-F modifications.

10. The nucleic acid according to claim 8, wherein the nucleosides of the first strand contain a 2'-sugar modification pattern, and 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 strand consists of 5 2'-F modifications.

11. The nucleic acid according to claim 8, wherein the nucleosides of the first strand contain a 2'-sugar modification pattern, and 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 strand consists of 7 2'-F modifications.

12. The nucleosides of the first strand contain the following (5'-3'): Me-F-(Me) 3 -X 1 -(Me) 7 -F-Me-F-(Me) 7 [wherein, X 1 is a heat destabilizing modification] The nucleic acid according to claim 9, which contains the 2'-sugar modification pattern.

13. The nucleosides of the first strand contain the following (5'-3'): Me-F-(Me) 3 -X 1 -Me-(F) 2 -(Me) 4 -F-Me-F-(Me) 7 [wherein, X 1 is a heat destabilizing modification] The nucleic acid according to claim 10, which contains the 2'-sugar modification pattern.

14. Two phosphorothioate nucleoside linkages are respectively present between three consecutive positions in the region near the 5'-end of the second strand, and the first phosphorothioate nucleoside linkage is present between the first base nucleoside and the adjacent second base nucleoside when read from the 5'-end in the region near the 5'-end of the second strand, and the second phosphorothioate nucleoside linkage is present between the second base nucleoside and the adjacent third base nucleoside in the region near the 5'-end of the second strand. The nucleic acid according to any one of claims 1 to 13.

15. Two phosphorothioate nucleoside linkages are respectively present between three consecutive positions in both the 5'-end region and the 3'-end region of the first strand, whereby the terminal nucleosides in the 5'-end region and the 3'-end region of the first strand are respectively attached to the 5'- and 3'-adjacent second last nucleosides by phosphorothioate nucleoside linkages, and the 5'- and 3'-second last nucleosides are respectively attached to the 5'- and 3'-adjacent third last nucleosides by phosphorothioate nucleoside linkages. The nucleic acid according to any one of claims 1 to 14.

16. A nucleic acid for inhibiting the expression of a target gene, comprising a first strand that is at least partially complementary to a part 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 double-stranded region having a length of at least 17 nucleosides, 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] including a 2'-sugar and abasic modification pattern, and the nucleosides of the first strand include a 2'-sugar modification pattern selected from one of the following (5'-3'): Me-F-(Me) 3 -X 1 -(Me) 7 -F-Me-F-(Me) 7 [wherein, X 1 is a heat destabilizing modification] Me-F-(Me) 3 -X 1 -Me-(F) 2 -(Me) 4 -F-Me-F-(Me) 7 [wherein X 1 is a thermal destabilizing modification] A nucleic acid.

17. A nucleic acid for inhibiting the expression of a target gene, comprising a first strand that is at least partially complementary to a part 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 double-stranded region having a length of at least 17 nucleosides, 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 a reverse abasic nucleoside, and (s) represents a phosphorothioate linkage] comprises a 2'-sugar and an abasic modification pattern of, and the nucleosides of the first strand are as follows (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 heat destabilizing modification] 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 heat destabilizing modification] a nucleic acid comprising a 2'-sugar modification pattern selected from one of the following.

18. 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 second last nucleoside in the 5'-terminal region of the second strand, and (a) the second last abasic nucleoside is connected to the adjacent first base nucleoside in the adjacent 5'-terminal vicinity region through a reverse nucleoside internucleoside linkage, (b) the reverse linkage is a 5-5' reverse linkage, and (c) the linkage between the terminal abasic nucleoside and the second last abasic nucleoside is 3'-5' when read towards the end including the terminal abasic nucleoside and the second last abasic nucleoside. The nucleic acid according to any one of claims 2 to 17.

19. The nucleic acid according to any one of claims 1 to 18, 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.

20. The ligand moiety 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 the nucleic acid through a linker The nucleic acid according to claim 19.

21. Structure: 【Chemical 1】 [wherein, R 1 is independently selected from the group consisting of hydrogen, methyl and ethyl each time it appears, R 2 is selected from the group consisting of hydrogen, hydroxy, -OC 1～3 alkyl, -C(=O)OC 1～3 alkyl, halo and nitro X 1 and X 2 is independently selected from the group consisting of methylene, oxygen and sulfur each time it appears, m is an integer from 1 to 6, n is an integer from 1 to 10, q, r, s, t, v are independently integers from 0 to 4, provided that (i) q and r cannot both be 0 simultaneously, and (ii) s, t and v cannot all be 0 simultaneously, Z is an oligonucleoside] The nucleic acid according to claim 20.

22. Structure: 【Chemical 2】 [wherein, r and s are independently integers selected from 1 to 16, Z is an oligonucleoside] The nucleic acid according to claim 20.

23. The nucleic acid according to any one of claims 1 to 22, wherein the nucleic acid is an siRNA oligonucleoside.

24. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 23 in combination with a pharmaceutically acceptable excipient or carrier.

25. The nucleic acid or pharmaceutical composition according to any one of claims 1 to 24 for use in therapy.

26. The nucleic acid or pharmaceutical composition according to any one of claims 1 to 25 for use in the prevention or treatment of a disease related to a hemostatic disorder, such as a disease related to a hemostatic disorder such as hemophilia.

27. The nucleic acid or pharmaceutical composition according to any one of claims 1 to 26 for use in the prevention or treatment of diabetes.

28. The nucleic acid or pharmaceutical composition according to any one of claims 1 to 27 for use in the prevention or treatment of cardiovascular diseases.

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