Inhibitor of expression and / or function

Abstract

The present invention relates to inhibitors, compositions containing the inhibitors, and their use in the treatment or prevention of diabetes.

Description

Technical Field

[0001] The present invention provides inhibitors such as nucleic acid compounds such as siRNA, which are suitable for therapeutic use. In addition, the present invention provides methods for making such compounds, as well as methods for using such compounds for treating various diseases and conditions.

Background Art

[0002] Inhibitors such as oligonucleoside / oligonucleotide compounds, which are inhibitors of gene expression and / or the expression or function of other targets such as lncRNA, can have important therapeutic applications in medicine. Oligonucleotides / oligonucleosides can be used to silence genes that are the cause of specific diseases. Gene silencing prevents the formation of proteins by inhibiting translation. Importantly, gene silencing agents are promising alternatives to traditional small organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleosides / oligonucleotides that prevent the formation of proteins by gene silencing.

[0003] In particular, a number of modified siRNA compounds for diagnostic and therapeutic purposes, 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 in the past 20 years.

[0004] The present invention relates to inhibitors such as oligomers, for example nucleic acids, for example oligonucleoside / oligonucleotide compounds, and their use in the treatment and / or prevention of diseases.

[0005] In particular, there is still a need for inhibitors suitable for assisting in the prevention and / or treatment of diseases such as type 2 diabetes.

[0006] A mutation in the B4GALT1 gene that results in serine at the position corresponding to position 352 of the full-length / mature B4GALT1 polypeptide has been identified as being associated with a reduced risk of coronary artery disease (see International Publication No. WO 2018 / 226560 pamphlet and Montasser et al., Science 374, pp. 1221-1227 (2021), Dec. 3, 2021). The use of siRNA that hybridizes to a sequence within the endogenous B4GALT1 gene and reduces the expression of the B4GALT1 polypeptide in a subject's cells has been proposed as a means for treating subjects who have developed or are susceptible to developing cardiovascular disease.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0007]

PATENT DOCUMENT 1

NON-PATENT DOCUMENTS

[0008]

NON-PATENT DOCUMENT 1

SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

[0009] The present invention is defined in the claims and relates, inter alia, to the following. In one aspect, the present invention relates to an inhibitor of post-translational glycosylation, such as an inhibitor of the expression and / or function of B4GALT1, which is conjugated to one or more ligand moieties.

[0010] In a further aspect, the present invention relates to an inhibitor according to the present invention, comprising an siRNA oligomer conjugated to one or more ligand moieties.

[0011] In a further aspect, the invention relates to an inhibitor according to the invention, wherein the one or more ligand moieties comprise one or more GalNAc ligands.

[0012] In a further aspect, the invention relates to an inhibitor according to the invention, wherein the one or more ligand moieties comprise another GalNAc ligand derivative.

[0013] In another aspect, the invention relates to an inhibitor of post-translational glycosylation for use in the treatment of diabetes, such as an inhibitor of the expression and / or function of B4GALT1.

[0014] In another aspect, the invention relates to an inhibitor of the expression and / or function of B4GALT1 for use in the treatment of diabetes.

[0015] In a further aspect, the invention relates to an inhibitor for use according to the invention, which is typically an siRNA oligomer conjugated to one or more ligand moieties.

[0016] In a further aspect, the invention relates to an inhibitor for use according to the invention, wherein the one or more ligand moieties comprise one or more GalNAc ligands and / or one or more GalNAc ligand derivatives.

[0017] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein the target of the inhibitor is selected from B4GALT1.

[0018] In a further aspect, the invention is an siRNA oligomer having a first strand and a second strand, i) the first strand of the siRNA has a length in the range of 15 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 23 or 25, even more preferably 23, and / or ii) The second strand of the siRNA has a length in the range of 15 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 21 nucleosides, which relates to an inhibitor according to the invention or an inhibitor for use according to the invention, which is an siRNA oligomer.

[0019] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein the second sense strand further comprises one or more abasic nucleosides in the terminal region of the second strand, and the abasic nucleosides are connected to adjacent nucleosides via inverse internucleoside linkages.

[0020] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein the second strand i two or more abasic nucleosides in the terminal region of the second strand, and / or ii two or more abasic nucleosides in either the 5' or 3' terminal region of the second strand, and / or iii two or more abasic nucleosides in either the 5' or 3' terminal region of the second strand, which are present in an overhang as described herein, and / or iv two or more consecutive abasic nucleosides in the terminal region of the second strand, preferably one such abasic nucleoside is the terminal nucleoside, two or more consecutive abasic nucleosides, and / or v two or more consecutive abasic nucleosides in either the 5' or 3' terminal region of the second strand, preferably one such abasic nucleoside is the terminal nucleoside of either the 5' or 3' terminal region of the second strand, two or more consecutive abasic nucleosides, and / or vi an inverse internucleoside linkage connecting at least one abasic nucleoside to an adjacent base nucleoside in the terminal region of the second strand, and / or vii At least one abasic nucleoside is connected to an adjacent base nucleoside by a reverse internucleoside linkage in either the 5' or 3' terminal region of the second strand, and / or viii An abasic nucleoside as the penultimate nucleoside connected via a reverse linkage to a nucleoside that is not the terminal nucleoside (referred to herein as the third last nucleoside), and / or ix An abasic nucleoside as two terminal nucleosides connected via a 5'-3' linkage when reading the strand in the direction towards its terminus, x An abasic nucleoside as two terminal nucleosides connected via a 3'-5' linkage when reading the strand in the direction towards the terminus including the terminal nucleoside, xi An abasic nucleoside at 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, xii An abasic nucleoside at the two terminal positions, wherein the penultimate nucleoside is connected to the third last nucleoside via a reverse linkage, (1) The reverse linkage is a 5-5' reverse linkage, and the linkage between the terminus and the penultimate abasic nucleoside is 3'5' when reading towards the terminus including the terminus and the penultimate abasic nucleoside, or (2) The reverse linkage is a 3-3' reverse linkage, and the linkage between the terminus and the penultimate abasic nucleoside is 5'3' when reading towards the terminus including the terminus and the penultimate abasic nucleoside, either of which is an abasic nucleoside relates to an inhibitor according to the invention or an inhibitor for use according to the invention, comprising the same.

[0021] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein the reverse internucleoside linkage is present in a terminal region distal from the 5' terminal region of the second strand or in a terminal region distal from the 3' terminal region of the second strand.

[0022] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the reverse nucleoside internucleoside linkage is a 3'3 reverse linkage.

[0023] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the reverse nucleoside internucleoside linkage is a 5'5 reverse linkage.

[0024] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein one or more nucleosides of the first strand and / or the second strand are modified to form modified nucleosides.

[0025] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the modification is a modification at the 2'-OH group of the ribose sugar and is optionally selected from a 2'-Me modification or a 2'-F modification.

[0026] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the first strand contains 2'-F at the 14th, 2nd, 6th position, or any combination thereof, counted from the 1st position of the first strand.

[0027] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the second strand contains a 2'-F modification at the 7th, and / or 9th, and / or 11th, and / or 13th position, counted from the 1st position of the second strand.

[0028] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the first strand and the second strand each contain a 2'-Me modification and a 2'-F modification.

[0029] In a further aspect, the invention relates to an siRNA, which preferably contains at least one heat destabilizing modification at one or more of positions 1 to 9 of the first strand, counting from position 1 of the first strand, and / or at one or more of the positions of the second strand aligned with positions 1 to 9 of the first strand. The destabilizing modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), and is preferably glycol nucleic acid, and relates to an inhibitor according to the invention or an inhibitor for use according to the invention.

[0030] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein the siRNA contains at least one heat destabilizing modification at position 7 of the first strand, counting from position 1 of the first strand.

[0031] In a further aspect, the invention relates to an siRNA, and the siRNA contains three or more 2'-F modifications at positions 7 to 13 of the second strand, counting from position 1 of the second strand, for example, 4, 5, 6, or 7 2'-F modifications at positions 7 to 13 of the second strand, and relates to an inhibitor according to the invention or an inhibitor for use according to the invention.

[0032] In a further aspect, the invention relates to an siRNA, and the second strand contains at least three, for example, 4, 5, or 6 2'-Me modifications at positions 1 to 6 of the second strand, counting from position 1 of the second strand, and relates to an inhibitor according to the invention or an inhibitor for use according to the invention.

[0033] In a further aspect, the invention relates to an siRNA, and the first strand preferably contains at least five consecutive 2'-Me modifications in the 3'-terminal region, including the terminal nucleoside of the 3'-terminal region, or within at least one or two nucleosides from the terminal nucleoside of the 3'-terminal region, and relates to an inhibitor according to the invention or an inhibitor for use according to the invention.

[0034] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, which is an siRNA, wherein the first strand preferably comprises seven consecutive 2'-Me modifications in the 3' terminal region, including the terminal nucleoside of the 3' terminal region.

[0035] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the siRNA oligomer further comprises one or more phosphorothioate internucleoside linkages.

[0036] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein each of the one or more phosphorothioate internucleoside linkages is present between at least three consecutive positions in the 5' or 3' proximal terminal region of the second strand, and the proximal terminal region is preferably adjacent to the terminal region where one or more abasic nucleosides of the second strand are located as defined herein.

[0037] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein each of the one or more phosphorothioate internucleoside linkages is present between at least three consecutive positions in the 5' and / or 3' terminal region of the first strand, and preferably, the terminal positions of the 5' and / or 3' terminal region of the first strand are attached to their adjacent positions by a phosphorothioate internucleoside linkage.

[0038] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the oligomer is an siRNA, and the second strand of the siRNA is directly or indirectly conjugated to one or more ligand moieties, and the ligand moiety is typically present in the terminal region of the second strand, preferably in its 3' terminal region.

[0039] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the ligand moiety is i) one or more GalNAc ligands, and / or ii) one or more GalNAc ligand derivatives, and / or iii) one or more GalNAc ligands and / or GalNAc ligand derivatives conjugated to said siRNA via a linker relates to an inhibitor according to the invention or an inhibitor for use.

[0040] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein said one or more GalNAc ligands and / or GalNAc ligand derivatives are conjugated directly or indirectly to the 5' or 3' terminal region, preferably the 3' terminal region, of the second strand of the siRNA oligomer.

[0041] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the ligand moiety

[0042]

Chemical formula

[0043] In a further aspect, the invention relates to a structure:

[0044]

Chemical formula

[0045] In a further aspect, the invention relates to a structure:

[0046]

Chemical formula

[0047] In a further aspect, the invention relates to an inhibitor according to the invention formulated as a pharmaceutical composition having excipients and / or carriers.

[0048] In another aspect, the invention relates to a pharmaceutical composition comprising an inhibitor according to the invention in combination with a pharmaceutically acceptable excipient or carrier.

[0049] In a further aspect, the invention relates to a pharmaceutical composition comprising an inhibitor according to the invention in combination with a pharmaceutically acceptable excipient or carrier for use in the treatment of diabetes.

[0050] In another aspect, the invention relates to the use of B4GALT1 as a target for identifying one or more therapeutic agents for treating diabetes.

[0051] In another aspect, the present invention relates to a method for treating or preventing diabetes, the method comprising administering to a patient an inhibitor of B4GALT1, such as an inhibitor defined according to the present invention, such as an inhibitor of post-translational glycosylation.

[0052] In another aspect, the present invention relates to B4GALT1 for use as a biomarker for diabetes.

[0053] In another aspect, the present invention relates to B4GALT1 for use in an in vivo method for predicting susceptibility to diabetes, typically by monitoring the level of the sequence and / or expression and / or function of B4GALT1 in a sample obtained from a patient.

[0054] In another aspect, the present invention is a method for predicting a patient's susceptibility to diabetes and optionally treating diabetes, the method comprising: (a) obtaining a sample from the patient; (b) detecting the sequence and / or expression and / or function of B4GALT1 in the sample obtained from the patient; (c) predicting susceptibility to diabetes based on the sequence and / or expression and / or function of B4GALT1 in the sample obtained from the patient; (d) preferably, administering an effective amount of a B4GALT1 inhibitor to the diagnosed patient. The present invention relates to a method comprising the above steps.

[0055] In another aspect, the present invention relates to the use of an inhibitor or composition according to the present invention in the preparation of a medicament for use in the treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0056]

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Mode for Carrying Out the Invention

[0057] The present invention provides inhibitors, such as oligomers such as nucleic acids, for example inhibitory RNA molecules (which may be referred to as iRNA or siRNA), and compositions containing them, which can affect the expression of a target, for example by binding to mRNA transcribed from a gene or by inhibiting the function of a nucleic acid such as long non-coding RNA (referred to herein as "lncRNA"). The target may be present within a cell, for example within a cell in a subject such as a human. The inhibitor can be used, for example, for the prevention and / or treatment of the expression of a target gene or a medical condition associated with the presence / activity of a nucleic acid in a cell such as long non-coding RNA.

[0058] In particular, the present invention identifies inhibitors of post-translational glycosylation, such as inhibitors of B4GALT1, which are useful for the prevention and / or treatment of diabetes.

[0059] B4GALT1 is beta-1,4-galactosyltransferase 1 and is an enzyme encoded by the B4GALT1 gene (SEQ ID NO: 1) in humans.

[0060] Genomic DNA sequence containing the B4GALT1 gene (SEQ ID NO: 1)

[0061] In particular, in the present invention, a large-scale GWAS meta-analysis (Mahajan et al., Nature Genetics, 2018, Vol. 50, pp. 1505-1513) in which 9% of 898,930 human individuals had diabetes was evaluated, and surprisingly, it was found that translational glycosylation was significantly associated with type 2 diabetes risk in both healthy and obese individuals. Importantly, the association between type 2 diabetes and translational glycosylation could not be identified by standard "functional enrichment" methods and was therefore not identified by the original authors of the meta-analysis.

[0062] Computational predictions were confirmed in in vivo mouse studies (Example 9). In these studies, mice were treated with siRNA that inhibits the expression of B4GALT1. In these mice, the plasma levels of LDL cholesterol, fasting blood glucose, and fibrinogen were significantly lower than those in untreated mice, suggesting that inhibition of B4GALT1 can lead to the prevention and / or treatment of diabetes, particularly type 2 diabetes.

[0063] Accordingly, the present invention relates to inhibitors of targets within the post-translational glycosylation pathway, such as enzymes involved in such pathways as B4GALT1. The inhibition may be of the gene or of the protein resulting from the expression of the gene, and reference to a gene such as B4GALT1 thereby expressly incorporates reference to inhibition of the expression or function of the gene and, separately, to inhibition of the protein product.

[0064] Post-translational glycosylation preferably refers to post-translational glycosylation seen in vivo in humans or human cells.

[0065] Definition The "first strand" is also referred to herein as the antisense strand or the guide strand, which can be used interchangeably herein, and refers to a nucleic acid strand, such as an siRNA strand, for example a dsiRNA strand, that contains a region that is substantially complementary to a target sequence, such as an mRNA. As used herein, the term "complementary region" refers to a region of the antisense strand that is substantially complementary to a sequence, such as a target sequence. If the complementary region is not completely complementary to the target sequence, the mismatch may be in the internal region or the terminal region of the molecule. In some embodiments, the double-stranded nucleic acid of the present invention, such as an siRNA agent, contains nucleotide mismatches in the antisense strand.

[0066] The "second strand" (also referred to herein as the sense strand or the passenger strand, which can be used interchangeably herein) refers to a nucleic acid strand, such as an siRNA strand, that contains a region that is substantially complementary to the region of the antisense strand as defined herein.

[0067] In the context of a molecule comprising a nucleic acid with a ligand moiety and optionally also a linker moiety, the nucleic acid of the present invention may be referred to as an oligonucleotide moiety or an oligonucleoside moiety.

[0068] 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 joined by such phosphodiester bonds between adjacent nucleosides, and other oligomers of nucleosides joined by bonds other than phosphate bonds are contemplated. For example, the bond between nucleotides may be a phosphorothioate bond. Thus, the term "oligonucleoside" herein encompasses both oligonucleotides and other oligomers of nucleosides. According to the present invention, 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 strand and / or the second strand) are also preferred.

[0069] In some embodiments, the double-stranded nucleic acids of the present invention, such as siRNA agents, contain nucleoside mismatches in the sense strand. In some embodiments, the nucleoside mismatch is present, for example, within 5, 4, 3, 2, or 1 nucleoside from the 3' end of the nucleic acid, such as siRNA.

[0070] In another embodiment, the nucleoside mismatch is present, for example, in the 3' terminal nucleoside of the nucleic acid, such as siRNA.

[0071] A "target sequence" (sometimes referred to as a target RNA or target mRNA) refers to a continuous portion of the nucleoside sequence of an mRNA molecule formed during the transcription of a gene, including the mRNA that is the product of RNA processing of the primary transcript, or may be a continuous portion of the nucleotide sequence of any RNA molecule, such as an lncRNA, that is desired to be inhibited.

[0072] 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 in length, 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 being part of the present invention.

[0073] The term "ribonucleoside" or "nucleoside" can also refer to a modified nucleoside, as will be described in more detail below.

[0074] The nucleic acid may be DNA or RNA and may contain modified nucleosides. A preferred nucleic acid is RNA.

[0075] As used interchangeably herein, the terms "iRNA", "siRNA", "RNAi agent", and "iRNA agent", "RNA interference agent" 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 by RNA interference (RNAi).

[0076] 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", and refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands that are said to have a "sense" direction and an "antisense" direction with respect to the target RNA. Most of the nucleosides of each strand of the nucleic acid, e.g., the dsiRNA molecule, are preferably ribonucleosides, but in that case, each strand or both strands may further contain one or more non-ribonucleosides, such as deoxyribonucleosides or modified ribonucleosides. In addition, as used herein, "siRNA" may contain ribonucleosides having chemical modifications.

[0077] The term "modified nucleoside" independently refers to a nucleoside having a modified sugar moiety, a modified internucleoside linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleoside includes substitutions, additions, or removals of, for example, functional groups or atoms, to the internucleoside linkage, sugar moiety, or nucleobase. Any such modifications used in siRNA-type molecules are included herein and in the claims for the purposes of "iRNA" or "RNAi agent" or "siRNA" or "siRNA agent".

[0078] The double-stranded region of the nucleic acid of the present invention, such as dsRNA, is about 9 to 40 base pairs in length, such as 9 to 36 base pairs in length, such as about 15 to 30 base pairs in length, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15 to 30, 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 base pairs in length may also be within the range.

[0079] The two strands forming the double-stranded structure may be different parts of one larger molecule or may be separate molecules, such as RNA molecules.

[0080] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the double-stranded structure of a double-stranded nucleic acid. The ds nucleic acid may contain an overhang of at least one nucleoside, or alternatively, the overhang may contain at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more nucleosides. The nucleoside overhang may contain or consist of a nucleoside analog containing a deoxynucleoside. The overhang may be present in the sense strand, the antisense strand, or any combination thereof. Further, the overhanging nucleosides may be present at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand.

[0081] In certain embodiments, the antisense strand has a 1 - 10 nucleoside overhang, for example, a 0 - 3, 1 - 3, 2 - 4, 2 - 5, 4 - 10, 5 - 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside overhang at the 3' or 5' end.

[0082] "Blunt" or "blunt end" means that there are no unpaired nucleosides at the ends of the double - stranded nucleic acid, i.e., there are no nucleoside overhangs. Nucleic acids of the present invention include those having no nucleoside overhang at one end or no nucleoside overhangs at either end.

[0083] Unless otherwise indicated, the term "complementary" is used to describe a first nucleoside sequence in relation to a second nucleoside sequence such that an oligonucleoside containing the first nucleoside sequence is capable of hybridizing under certain conditions to an oligonucleoside or polynucleotide 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).

[0084] The complementary sequences in the nucleic acids described herein, e.g., in dsiRNA, involve base pairing over the entire length of one or both nucleoside sequences of an oligonucleoside or polynucleotide containing a first nucleoside sequence and an oligonucleoside or polynucleotide containing a second nucleoside sequence. Such sequences can be referred to herein as "fully complementary" to each other. However, herein, when a first sequence is referred to as "substantially complementary" or "partially complementary" to a second sequence, the two sequences may be fully complementary or form one or more, but preferably no more than 5 mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, while retaining the ability to hybridize under conditions most relevant to the ultimate application, e.g., inhibition of gene expression via the RISC pathway. Bulges shall not be considered mismatches with respect to the determination of complementarity. Further, for example, a nucleic acid, e.g., dsRNA, 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 be called "fully complementary".

[0085] Also, "complementary" sequences, as used herein, may or may only contain base pairs formed from non-Watson-Crick base pairs or non-natural and modified nucleosides, as long as they meet the above requirements regarding their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogsteen base pairing.

[0086] The terms "complementary", "fully complementary", and "substantially / partially complementary" can be used herein with respect to base matching between the sense and antisense strands of a nucleic acid, e.g., dsiRNA, or between the antisense strand of a double-stranded nucleic acid, e.g., siRNA agent, and a target sequence.

[0087] Within the present invention, the second strand of the nucleic acid according to the present invention, particularly the dsiRNA for inhibiting B4GALT1, is at least partially complementary to the first strand of the nucleic acid. In certain embodiments, the first and second strands of the nucleic acid according to the present invention have a length of at least 17 base pairs and are partially complementary when forming a double-stranded region containing 1, 2, 3, 4, or 5 or fewer mismatched base pairs.

[0088] In certain embodiments, the first and second strands of the nucleic acid according to the present invention have a length of 19 base pairs and are partially complementary when forming a double-stranded region having 1, 2, 3, 4, or 5 or fewer mismatched base pairs. In certain embodiments, the first and second strands of the nucleic acid according to the present invention have a length of 21 base pairs and are partially complementary when forming a double-stranded region having 1, 2, 3, 4, or 5 or fewer mismatched base pairs.

[0089] Alternatively, the first and second strands of the nucleic acid according to the present invention are a double-stranded region having a length of at least 17 base pairs, wherein at least 14, 15, 16, or 17 of the base pairs are complementary base pairs, particularly Watson-Crick base pairs, and are partially complementary when forming a double-stranded region.

[0090] In certain embodiments, the first and second strands of the nucleic acid according to the present invention are a double-stranded region having a length of 19 base pairs, wherein all at least 14, 15, 16, 17, 18, or 19 base pairs are complementary base pairs, particularly Watson-Crick base pairs, and are partially complementary when forming a double-stranded region. In certain embodiments, the first and second strands of the nucleic acid according to the present invention are a double-stranded region having a length of 21 base pairs, wherein all at least 16, 17, 18, 19, 20, or 21 base pairs are complementary base pairs, particularly Watson-Crick base pairs, and are partially complementary when forming a double-stranded region.

[0091] 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 polynucleotide that is substantially or partially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a gene). In certain embodiments, the contiguous portion of the mRNA is any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201. For example, a polynucleotide is complementary to at least a portion of the mRNA of a gene of interest if its sequence is substantially or partially complementary to an uninterrupted portion of the mRNA encoding that gene.

[0092] Accordingly, in some preferred embodiments, the antisense oligonucleotides disclosed herein are fully complementary to the target gene sequence.

[0093] In other embodiments, the antisense oligonucleotides disclosed herein are substantially or partially complementary to the target RNA sequence and contain a contiguous nucleoside sequence that is at least about 80% complementary, e.g., at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, over its entire length to the equivalent region of the target RNA sequence.

[0094] In certain embodiments, the first (antisense) strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of the RNA transcribed from the B4GALT1 gene. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of at least 17 nucleosides of B4GALT1 mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleosides of B4GALT1 mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of 17, 18, or 19 nucleosides of any one of the sequences listed in Table 1, namely any one of SEQ ID NOs: 2-21 or 102-201. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of 19, 20, 21, 22, or 23 nucleosides of any one of SEQ ID NOs: 102-201.

[0095] In certain embodiments, the first (antisense) strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of at least 17 nucleosides, and at least 14, 15, 16, or 17 nucleosides of said continuous nucleoside sequence are partially complementary to a continuous portion of the B4GALT1 mRNA when they are complementary to a continuous portion of the B4GALT1 mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of at least 17 nucleosides, and at least 14, 15, 16, or 17 nucleosides of said continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201. In certain embodiments, the first strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of 19 nucleosides, and at least 14, 15, 16, 17, 18, or all 19 nucleosides of said continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201. In certain embodiments, the first strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of 21 nucleosides, and at least 16, 17, 18, 19, 20, or all 21 nucleosides of said continuous nucleoside sequence are complementary to a continuous portion of any one of SEQ ID NOs: 102-201. In certain embodiments, the first strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of 23 nucleosides, and at least 18, 19, 20, 21, 22, or all 23 nucleosides of said continuous nucleoside sequence are complementary to a continuous portion of any one of SEQ ID NOs: 102-201.

[0096] 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 polynucleotide, and the antisense polynucleotide is, in turn, complementary to a target gene sequence and is at least about 80% complementary over its entire length to an equivalent region of the nucleotide sequence of the antisense strand, 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 continuous nucleotide sequence.

[0097] 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 is at least 80% complementary over its entire length to the target sequence, 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 continuous nucleotide sequence.

[0098] As used herein, a "subject" is an animal, including a mammal such as a primate (human, non-human primate such as a monkey and chimpanzee, etc.) or non-primate, or a bird, that expresses the target gene either endogenously or heterologously when the target gene sequence has sufficient complementarity to promote target knockdown against a nucleic acid, such as an iRNA agent. In certain preferred embodiments, the subject is human.

[0099] The terms "treat" or "treatment" refer to beneficial or desired results, including, but not limited to, alleviation or amelioration of one or more symptoms associated with gene expression. "Treatment" may mean extending the survival period as compared to the expected survival period without treatment. Treatment may include prevention of the onset of complications, e.g., reduction of liver damage in a subject having a liver infection.

[0100] As used herein, "therapeutically effective amount" is intended to include an amount of a nucleic acid, such as an iRNA, that is sufficient to achieve treatment of a disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms or associated complications thereof) when administered to a patient for treating a subject having the disease.

[0101] As used herein, the term "pharmaceutically acceptable" is used to refer to compounds, substances, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without undue toxicity, irritation, allergic response, or other problems or complications and that are commensurate with a reasonable benefit / risk ratio.

[0102] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, involved in carrying or transporting a subject compound from one organ or part of the body to another organ or part of the body. 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.

[0103] Where values or ranges of values of parameters are recited, intermediate values and ranges within the recited values are also intended to be part of the present invention.

[0104] As used herein, the articles "a" and "an" are used to refer to one or more than one (i.e., at least one) of the grammatical objects of the article.

[0105] As used herein, the term "comprising" is used so as to mean "comprising but not limited to" and is used interchangeably therewith.

[0106] As used herein, the term "or" means 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".

[0107] The term "about" is used herein to mean within the typical error tolerance ranges 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%. It is understood that when "about" is present before a series of numbers or a range, "about" can modify each of the numbers in that series of numbers or range.

[0108] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that can be logically included as apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides of a 21-nucleoside nucleic acid molecule" means that 18, 19, 20, or 21 nucleosides have the stated characteristics. It is understood that when "at least" is present before a series of numbers or a range, "at least" can modify each of the numbers in that series of numbers or range.

[0109] As used herein, "below" or "less than" is understood to mean the value adjacent to this phrase, and, in theory, lower values, and in some cases, logically zero values or integers. For example, a duplex having an overhang "of 2 nucleosides or less" has an overhang of 2, 1, or 0 nucleosides. It is understood that when "below" is present after a series of numbers or a range, "below" can modify each of the numbers in that series of numbers or range.

[0110] The terminal region of a strand is the last 5 nucleotides from the 5' or 3' end.

[0111] The nucleobase sequence is the sequence of bases of an oligomeric nucleic acid.

[0112] The various embodiments of the present invention can be combined as determined to be appropriate by those skilled in the art.

[0113] Target Targets for inhibition disclosed herein may be, but are not limited to, mRNA, lncRNA, polypeptide, protein, or gene.

[0114] Targets are targets involved in the post-translational glycosylation pathway of proteins herein. These are preferably targets whose inhibition aids in the prevention or treatment of diabetes. A preferred target for inhibition is B4GALT1, and inhibition can be achieved by inhibiting the expression or function of the gene or protein or both.

[0115] In one aspect, the target is mRNA or long non-coding RNA (lncRNA) expressed from a gene.

[0116] In a preferred embodiment, the target is mRNA that is a result of the expression of the B4GALT1 gene. Exemplary target sequences of B4GALT1 mRNA are listed in Table 1 below.

[0117] [Table 1] JPEG2025519286000005.jpg252167JPEG2025519286000006.jpg100167

[0118] It should be understood that SEQ ID NOs: 2 to 21 and 102 to 201 relate to human (Homo sapiens) mRNA sequences.

[0119] Disease / condition The present invention relates to inhibitors suitable for or for use in the treatment of diabetes such as type 1 diabetes or type 2 diabetes, preferably type 2 diabetes.

[0120] Inhibitor Examples of the inhibitor of the present invention include nucleic acids such as siRNA, antibodies and their antigen-binding fragments, for example, monoclonal antibodies, polypeptides, antibody-drug conjugates, and small molecules. Nucleic acids such as siRNA are preferred.

[0121] Certain preferred features of the inhibitor of the present invention, which is an oligonucleoside such as siRNA, are shown below.

[0122] In certain embodiments, the nucleic acid comprises a first strand comprising a sequence at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene (SEQ ID NO: 1). In a preferred embodiment, the nucleic acid comprises a first strand comprising a sequence at least partially complementary to B4GALT1 mRNA (NM_001497.4).

[0123] In certain embodiments, the nucleic acid for inhibiting the expression of B4GALT1 comprises a double-stranded region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand (i) is at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, (ii) comprises at least 17 consecutive nucleosides that differ from any one of SEQ ID NOs: 22-41 or 202-301 by 0 or 1 nucleoside.

[0124] In certain embodiments, the first strand comprises nucleosides 2-18 of any one of the sequences shown in SEQ ID NOs: 22-41 or 202-301.

[0125] In certain embodiments, the nucleic acid for inhibiting the expression of B4GALT1 comprises a double-stranded region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand (i) It is at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, (ii) It contains at least 21 consecutive nucleosides that differ from any one of SEQ ID NOs: 202 to 301 by 0 or 1 nucleoside.

[0126] In certain embodiments, the first strand comprises nucleosides 2 to 22 of any one of the sequences shown in SEQ ID NOs: 202 to 301.

[0127] In certain embodiments, the first strand comprises any one of SEQ ID NOs: 22 to 41 or 202 to 301.

[0128] In certain embodiments, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of SEQ ID NOs: 42 to 61 or 302 to 401 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleosides.

[0129] In certain embodiments, the second strand comprises a nucleoside sequence of at least 19 consecutive nucleosides that differ from any one of SEQ ID NOs: 302 to 401 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 19 consecutive nucleosides.

[0130] In certain embodiments, the second strand comprises a nucleoside sequence of at least 21 consecutive nucleosides that differ from any one of SEQ ID NOs: 302 to 401 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 21 consecutive nucleosides.

[0131] In certain embodiments, the second strand comprises any one of SEQ ID NOs: 42 to 61 or 302 to 401.

[0132] In certain embodiments, the nucleic acid comprises, consists of, or consists essentially of a first strand having a nucleoside sequence that differs from any one of SEQ ID NOs: 22-41 or 202-301 by 0 or 1 nucleoside, and a second strand having a nucleoside sequence that differs from any one of SEQ ID NOs: 42-61 or 302-401 by 0 or 1 nucleoside.

[0133] In this specification, the double-stranded region is preferably formed between a first (antisense) strand and a complementary second (sense) strand. Exemplary pairs of complementary antisense and sense strands are listed in Table 2 below.

[0134] [Table 2] JPEG2025519286000008.jpg251167JPEG2025519286000009.jpg251167JPEG2025519286000010.jpg251167JPEG2025519286000011.jpg110167

[0135] In certain embodiments, the invention relates to a nucleic acid comprising, consisting of, or consisting essentially of a first strand and a second strand having a nucleoside sequence that differs from any one of the following first and second sequences by 0 or 1 nucleoside.

[0136] [Table 3]

[0137] In certain embodiments, the nucleic acid for inhibiting the expression of B4GALT1 comprises a double-stranded region comprising a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand (i) is at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, (ii) It contains at least 17 consecutive nucleosides that differ from any one of SEQ ID NOs: 62 to 81 or 402 to 513 by 0 or 1 nucleoside.

[0138] In certain embodiments, the first strand contains nucleosides 2 to 18 of any one of the sequences shown in SEQ ID NOs: 62 to 81 or 402 to 513.

[0139] In certain embodiments, the nucleic acid for inhibiting the expression of B4GALT1 contains a double-stranded region including a first strand and a second strand that is at least partially complementary to the first strand, and the first strand (i) is at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, (ii) contains at least 21 consecutive nucleosides that differ from any one of SEQ ID NOs: 402 to 513 by 0 or 1 nucleoside.

[0140] In certain embodiments, the first strand contains nucleosides 2 to 22 of any one of the sequences shown in SEQ ID NOs: 402 to 513.

[0141] In certain embodiments, the first strand contains any one of SEQ ID NOs: 62 to 81 or 402 to 513.

[0142] The modification patterns of the nucleic acids shown in SEQ ID NOs: 62 to 81 and 402 to 513 are summarized in Table 3 below.

[0143]

Table 4

[0144] In certain embodiments, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs from any one of SEQ ID NOs: 82-101 or 514-621 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleosides.

[0145] In certain embodiments, the second strand comprises a nucleoside sequence of at least 19 consecutive nucleosides that differs from any one of SEQ ID NOs: 514-621 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 19 consecutive nucleosides.

[0146] In certain embodiments, the second strand comprises a nucleoside sequence of at least 21 consecutive nucleosides that differs from any one of SEQ ID NOs: 514-621 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 21 consecutive nucleosides.

[0147] In certain embodiments, the second strand comprises any one of SEQ ID NOs: 82-101 or 514-621.

[0148] The modification patterns of the nucleic acids shown in SEQ ID NOs: 82-101 and 514-621 are summarized in Table 4 below.

[0149]

Table 5

[0150] As used herein, particularly in Tables 3 and 4, the following abbreviations are used with respect to modified nucleosides.

[0151] Am represents 2'-O-methyl-adenosine, Cm represents 2'-O-methyl-cytidine, Gm represents 2'-O-methyl-guanosine, Um represents 2'-O-methyl-uridine, Af represents 2'-fluoro-adenosine, Cf represents 2'-fluoro-cytidine, Gf represents 2'-fluoro-guanosine, and Uf represents 2'-fluoro-uridine.

[0152] Furthermore, the letter "s" is used as an abbreviation for a phosphorothioate linkage between two consecutive (modified) nucleosides. For example, the abbreviation "AmsAm" is used for two consecutive 2'-O-methyl-adenosine nucleosides linked via a 3'-5' phosphorothioate linkage. No abbreviation is used for nucleosides linked via a standard 3'-5' phosphodiester linkage. For example, the abbreviation "AmAm" is used for two consecutive 2'-O-methyl-adenosine nucleosides linked via a 3'-5' phosphodiester linkage.

[0153] In certain embodiments, the nucleic acid comprises, consists of, or consists essentially of a first strand having a (modified) nucleoside sequence that differs from any one of SEQ ID NOs: 62-81 or 402-513 by 0 or 1 nucleoside, and a second strand having a (modified) nucleoside sequence that differs from any one of SEQ ID NOs: 82-101 or 514-621 by 0 or 1 nucleoside.

[0154] Preferred combinations of complementary modified antisense (first) and sense (second) strands are listed in Table 5 below.

[0155]

Table 6

[0156] In a particularly preferred embodiment, the invention relates to a nucleic acid comprising, consisting of, or consisting essentially of a first strand and a second strand having a nucleoside sequence that differs from either one of the following first and second sequences by 0 or 1 nucleoside.

[0157]

Table 7

[0158] If there is any ambiguity between the sequences in this specification and the sequences in the attached sequence listing, the sequences provided in this specification shall be regarded as the correct sequences.

[0159] Apurinic / apyrimidinic nucleotide In certain embodiments, the nucleic acid according to the invention has one, for example two, for example three, for example four, or more apurinic / apyrimidinic nucleosides. Apurinic / apyrimidinic nucleosides are modified nucleosides because they lack the base normally found at the 1-position of the sugar moiety. Typically, there will be a hydrogen at the 1-position of the sugar moiety of the apurinic / apyrimidinic nucleosides present in the nucleic acid according to the invention.

[0160] Apurinic / apyrimidinic nucleosides are present in the terminal region of the second strand and are preferably located within the terminal 5 nucleosides at the end of the strand. The terminal region may be the terminal 5 nucleosides including the apurinic / apyrimidinic nucleoside.

[0161] The second strand may include the following preferred features (all combinations are specifically contemplated unless mutually exclusive): Two or more apurinic / apyrimidinic nucleosides in the terminal region of the second strand, and / or Two or more apurinic / apyrimidinic nucleosides in either the 5' or 3' terminal region of the second strand, and / or Two or more apurinic / apyrimidinic nucleosides in either the 5' or 3' terminal region of the second strand, which are present in the overhang as described herein, and / or Two or more consecutive abasic nucleosides in the terminal region of the second strand, preferably one such abasic nucleoside is the terminal nucleoside, two or more consecutive abasic nucleosides, and / or Two or more consecutive abasic nucleosides in either the 5’ or 3’ terminal region of the second strand, preferably one such abasic nucleoside is the terminal nucleoside of either the 5’ or 3’ terminal region of the second strand, two or more consecutive abasic nucleosides, and / or Inverse internucleoside linkages connecting at least one abasic nucleoside to an adjacent nucleoside in the terminal region of the second strand, and / or Inverse internucleoside linkages connecting at least one abasic nucleoside to an adjacent nucleoside in either the 5’ or 3’ terminal region of the second strand, and / or An abasic nucleoside as the penultimate nucleoside connected via an inverse linkage to a nucleoside that is not the terminal nucleoside (referred to herein as the third nucleoside from the end), and / or An abasic nucleoside as two terminal nucleosides connected via a 5’-3’ linkage when reading the strand in the direction towards the end including the terminal nucleoside, An abasic nucleoside as two terminal nucleosides connected via a 3’-5’ linkage when reading the strand in the direction towards the end including the terminal nucleoside, Abasic nucleosides at the last two positions of the end, wherein the penultimate nucleoside is connected to the third nucleoside from the end via an inverse linkage, and the inverse linkage is a 5-5’ inverse linkage or a 3’-3’ inverse linkage, abasic nucleosides, Abasic nucleosides at the last two positions of the end, wherein the penultimate nucleoside is connected to the third nucleoside from the end via an inverse linkage, (1) The reverse linkage is a 5-5' reverse linkage, and the linkage between the terminus and the second-to-last abasic nucleoside is 3'5' when read towards the terminus containing the terminus and the second-to-last abasic nucleoside, or (2) The reverse linkage is a 3-3' reverse linkage, and the linkage between the terminus and the second-to-last abasic nucleoside is either 5'3' when read towards the terminus containing the terminus and the second-to-last abasic nucleoside. An abasic nucleoside.

[0162] Preferably, an abasic nucleoside is present at the terminus of the second strand.

[0163] Preferably, in the terminal region of the second strand, preferably at the terminus and the second-to-last position, two or at least two abasic nucleosides are present.

[0164] Preferably, two or more abasic nucleosides are consecutive, for example, all abasic nucleosides may be consecutive. For example, the terminal 1 nucleotide or terminal 2 nucleotides or terminal 3 nucleotides or terminal 4 nucleotides may be abasic nucleosides.

[0165] Also, except when only one abasic nucleoside is present at the terminus, the abasic nucleoside may be linked to an adjacent nucleoside via a 5'-3' phosphodiester linkage or a reverse linkage. When only one abasic nucleoside is present, it will have a reverse linkage with the adjacent nucleoside.

[0166] The reverse linkage (which may also be called an 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.

[0167] Non-terminal abasic nucleosides will each have two phosphodiester linkages, one to each adjacent nucleoside, which may be reverse linkages, or 5'-3' phosphodiester linkages, or one of each.

[0168] A preferred embodiment includes two abasic nucleosides at the terminus and the penultimate position of the second strand, with the reverse internucleoside linkage located between the penultimate (abasic) nucleoside and the antepenultimate nucleoside.

[0169] Preferably, two abasic nucleosides are present at the terminus and the penultimate position of the second strand, with the penultimate nucleoside linked to the antepenultimate nucleoside via a reverse internucleoside linkage and to the terminal nucleoside via a 5'-3' or 3'-5' phosphodiester linkage (when read in the direction of the terminus of the molecule).

[0170] Various preferred features are as follows. The reverse internucleoside linkage is a 3'-3' reverse linkage. The reverse internucleoside linkage is present in the terminal region distal from the 5'-terminal phosphate of the second strand.

[0171] The reverse internucleoside linkage is a 5'-5' reverse linkage. The reverse internucleoside linkage is present in the terminal region distal from the 3'-terminal hydroxide of the second strand.

[0172] In certain embodiments, the second strand comprises two consecutive abasic nucleosides in the 5' terminal region of the second strand, one such abasic nucleoside being the terminal nucleoside of the 5' terminal region of the second strand and the other abasic nucleoside being the penultimate nucleoside of the 5' terminal region of the second strand, (a) the penultimate abasic nucleoside is linked to the adjacent first base nucleoside of the adjacent 5' proximal region via an inverse internucleoside linkage, (b) the inverse linkage is a 5-5' inverse linkage, and (c) the linkage between the terminal and penultimate abasic nucleosides is 3'-5' when read towards the terminus containing the terminal and penultimate abasic nucleosides. More typically, (i) the first and second strands each have a length of 19 or 23 nucleosides, (ii) two phosphorothioate internucleoside linkages are present between three consecutive positions in the 5' proximal region of the second strand, the first phosphorothioate internucleoside linkage is present between the adjacent first base nucleoside of (a) and the adjacent second base nucleoside of the 5' proximal region of the second strand, the second phosphorothioate internucleoside linkage is present between the adjacent second base nucleoside and the adjacent third base nucleoside of the 5' proximal region of the second strand, (iii) two phosphorothioate internucleoside linkages are present between three consecutive positions in both the 5' and 3' terminal regions of the first strand, the terminal nucleosides in each of the 5' and 3' terminal regions of the first strand are each attached by a phosphorothioate internucleoside linkage to the adjacent penultimate nucleoside at 5' and 3', respectively, and each of the first 5' and 3' penultimate nucleosides is attached by a phosphorothioate internucleoside linkage to the adjacent antepenultimate nucleoside at 5' and 3', respectively, and (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.

[0173] Alternatively, the second strand preferably includes two consecutive abasic nucleosides in the overhang of the 3' terminal region of the second strand, one such abasic nucleoside being the terminal nucleoside of the 3' terminal region of the second strand and the other abasic nucleoside being the penultimate nucleoside of the 3' terminal region of the second strand, (a) said penultimate abasic nucleoside being connected via an inverted internucleoside linkage to the adjacent first base nucleoside of the adjacent 3' proximal region, (b) the inverted linkage being a 3-3' inverted linkage, and (c) the linkage between the terminal and the penultimate abasic nucleoside being 5'-3' when read towards the terminus including the terminal and the penultimate abasic nucleoside. More typically, (i) the first strand and the second strand each have a length of 19 or 23 nucleosides, (ii) two phosphorothioate internucleoside linkages are present between three consecutive positions in said 3' proximal region of the second strand, the first phosphorothioate internucleoside linkage being present between (a) said adjacent first base nucleoside and the adjacent second base nucleoside of said 3' proximal region of the second strand, and the second phosphorothioate internucleoside linkage being present between said adjacent second base nucleoside and the adjacent third base nucleoside of said 3' proximal region of the second strand, (iii) two phosphorothioate internucleoside linkages are present between three consecutive positions in both the 5' and 3' terminal regions of the first strand, the terminal nucleosides in each of said 5' and 3' terminal regions of the first strand each being attached by a phosphorothioate internucleoside linkage to the adjacent penultimate nucleoside at 5' and 3', respectively, and each of the first 5' and 3' penultimate nucleosides being attached by a phosphorothioate internucleoside linkage to the adjacent antepenultimate nucleoside at 5' and 3', respectively, and (iv) the second strand of the nucleic acid is directly or indirectly conjugated to one or more ligand moieties in the 5' terminal region of the second strand.

[0174] Examples of such structures are as follows (the specific RNA nucleosides shown are not limiting and may be any RNA nucleoside). A 3’-3’ inverse linkage (and the last phosphodiester bond between two abasic molecules in the 5’-3’ direction when read towards the end of the molecule is also shown)

[0175]

Chem.

[0176]

Chem.

[0177] One or more abasic nucleosides present in the nucleic acid are provided in the presence of one or more inverse nucleoside linkages, i.e., 5’-5’ or 3’-3’ inverse nucleoside linkages. The inverse linkage results from a change in the orientation of the adjacent nucleoside sugars, such that the sugars will have a 3’-5’ orientation as opposed to the conventional 5’-3’ orientation (based on the numbering of the ring atoms of the nucleoside sugar). One or more abasic nucleosides present in the nucleic acids of the invention preferably contain such inverted nucleoside sugars.

[0178] When the terminal nucleoside has an inverted orientation, this will result in an "inversion" of the terminal configuration of the entire nucleic acid. Certain structures illustrated and referred to herein are represented using the conventional 5'-3' direction (based on the numbering of the ring atoms of the nucleoside sugar), but the change in orientation and the presence of a terminal nucleoside with a proximal 3'-3' inverse linkage will result in a nucleic acid having an overall 5'-5' terminal structure (i.e., the conventional 3' terminal nucleoside becomes the 5' terminal nucleoside), which will be understood. Alternatively, it will be understood that the change in orientation and the presence of a terminal nucleoside with a proximal 5'-5' inverse linkage will result in a nucleic acid having an overall 3'-3' terminal structure.

[0179] The proximal 3'-3' or 5'-5' inverse linkages described herein may include inverse linkages that are directly adjacent / attached to a terminal nucleoside having an inverted orientation, such as a single terminal nucleoside having an inverted orientation. Alternatively, the proximal 3'-3' or 5'-5' inverse linkages described herein may include inverse linkages that are adjacent to two or more nucleosides having an inverted orientation, such as two or more terminal region nucleosides having an inverted orientation, such as the terminal and the second last nucleoside. Thus, the inverse linkage may be attached to the second last nucleoside having an inverted orientation. One skilled in the art will understand that the inverted orientation described above can result in a nucleic acid molecule having the overall 3'-3' or 5'-5' terminal structure described herein, but when one or more additional inverse linkages and / or nucleosides having an inverted orientation are present, it will also be understood that the overall nucleic acid can have a 3'-5' terminal structure corresponding to the 5' / 3' terminal of the conventional configuration.

[0180] In one aspect, the nucleic acid may have a 3'-3' inverse 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.

[0181] Accordingly, one of ordinary skill in the art will clearly understand that 5'-5', 3'-3', and 3'-5' (read in the direction of their termini) terminal variants of the more general 5'-3' structure (based on the numbering of the ring atoms of the terminal nucleoside sugar) depicted herein are within the scope of the present disclosure if one or more inverse linkages are present.

[0182] For example, in the context of one or more nucleosides having an inverse orientation that creates an inverse internucleoside linkage and / or an inverse terminus, when the relative position of the linkage (e.g., relative to the linker) or the position of internal features (e.g., modified nucleosides) 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 inverse linkage were not arranged, and the conventional 5' or 3' terminus is determined by considering the majority of the directionality of the internal nucleoside linkages and / or nucleoside orientations within the nucleic acid. From such internal linkages and / or nucleoside orientations, it is possible to determine which terminus of the nucleic acid constitutes the conventional 5' and 3' termini (based on the numbering of the ring atoms of the terminal nucleoside sugar) of the molecule in the absence of the inverse linkage.

[0183] 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 inverse orientation, the "5'" terminus, which is the conventional 5' terminus shown in the figure below, can actually contain a 3'OH in light of the inverted nucleoside at the terminal position. Nevertheless, when read in the standard 5'[PO4] to 3'[OH] direction of the nucleic acid molecule (based on the numbering of the ring atoms of the nucleoside sugar), most of the molecule will contain conventional internucleoside linkages extending from the 3'OH of the sugar to the 5' phosphate of the next sugar, and these can be used to determine the conventional 5' and 3' termini where the absence of an inverted terminus configuration is found. A 5’A-A-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me3’

[0184] Inverse conjugation is preferably located at the end of a nucleic acid, such as RNA, distal to the ligand portion of the molecule, such as the GalNAc-containing portion.

[0185] A GalNAc-siRNA construct having 5'-GalNAc on the sense strand can have an inverse conjugation at the end opposite to the sense strand.

[0186] A GalNAc-siRNA construct having 3'-GalNAc on the sense strand can have an inverse conjugation at the end opposite to the sense strand.

[0187] Length of the nucleic acid In one aspect, i) the first strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides, and / or ii) the second strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0188] 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 the portion of the RNA transcribed from the B4GALT1 gene is 17 to 30 nucleosides in length.

[0189] In one aspect, i) the first strand of the nucleic acid has a length in the range of 15 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 23 or 25, and / or ii) the second strand of the nucleic acid has a length in the range of 15 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 23.

[0190] Generally, the double-stranded structure of nucleic acids, such as iRNA, is about 15 to 30 base pairs in length, for example 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 base pairs in length. Ranges and lengths intermediate to those described above are also contemplated as being part of the present invention.

[0191] Similarly, the region of complementarity of the antisense sequence to the target sequence and / or the region of complementarity of the antisense sequence to the sense sequence is about 15 to 30 nucleosides in length, for example 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 described above are also contemplated as being part of the present invention.

[0192] In certain preferred embodiments, the region of complementarity of the antisense sequence to the target sequence and / or the region of complementarity of the antisense sequence to the sense sequence is at least 17 nucleosides in length. For example, the region of complementarity between the antisense strand and the target is 19 to 21 nucleosides in length, for example, the region of complementarity is 21 nucleosides in length.

[0193] In preferred embodiments, each strand is 30 nucleosides or less in length.

[0194] In certain embodiments, the double-stranded structure of the nucleic acid, such as siRNA, is 19 base pairs in length. In particularly preferred embodiments, the double strand may have the following structure.

[0195]

Chemical formula

[0196] The nucleic acids described herein, such as dsRNA, may further comprise one or more single-stranded nucleoside overhangs, such as 1 to 4, 2 to 4, 1 to 3, 2 to 3, 1, 2, 3, or 4 nucleosides. The nucleoside overhangs may comprise or consist of nucleosides / nucleoside analogs comprising deoxynucleosides / nucleosides. The overhangs may be present in the sense strand, the antisense strand, or any combination thereof. Further, the nucleosides of the overhang may be present at the 5′ end, 3′ end, or both ends of the antisense or sense strand of the nucleic acid, such as dsRNA.

[0197] In certain preferred embodiments, at least one strand comprises a 3′ overhang of at least one nucleoside, for example, at least one strand comprises a 3′ overhang of at least two nucleosides. The overhang is preferably present in the antisense / guide strand and / or the sense / passenger strand.

[0198] 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.

[0199] 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 properties.

[0200] In certain embodiments of the invention, substantially all of the nucleosides are modified.

[0201] 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. This document is hereby incorporated herein by reference.

[0202] Modifications include terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, inverted ligation) or 3' terminal modifications (conjugation, DNA nucleosides in RNA, or RNA nucleosides in DNA, inverted ligation, etc.); base modifications, such as stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, substitution with conjugate bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; or backbone modifications, such as modifications or substitutions of the phosphodiester linkage.

[0203] Specific examples of nucleic acids such as siRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain a modified backbone or do not contain a native internucleoside linkage. Nucleic acids such as RNAs with a modified backbone include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification and sometimes as referred to in the art, modified nucleic acids that do not have a phosphorus atom in the internucleoside backbone, such as RNAs, can also be considered oligonucleosides. In some embodiments, the modified nucleic acid, such as siRNA, will have a phosphorus atom in its internucleoside backbone.

[0204] Modified nucleic acid backbones, such as for RNA, include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and the normal 3'-5' linkages, boranophosphates having their 2'-5' linkage analogs, and those having reverse polarity with adjacent pairs of nucleoside units linked 5'-3' or 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0205] In addition, modified nucleic acids, such as RNA, may 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 or may not be substituted. 2'-O-methyl and 2'-F are preferred modifications.

[0206] In certain preferred embodiments, the nucleic acid comprises at least one modified nucleoside.

[0207] The nucleic acids of the invention may comprise one or more modified nucleosides in the first strand and / or the second strand.

[0208] In some embodiments, substantially all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise modifications.

[0209] In some embodiments, all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand comprise modifications.

[0210] In some embodiments, all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand comprise modifications.

[0211] In one embodiment, at least one of the modified nucleosides is selected from the group consisting of: deoxy-nucleosides, 3'-terminal deoxy-thymidine (dT) nucleosides, 2'-O-methyl modified nucleosides (also referred to herein as 2'-Me, where Me is methoxy), 2'-fluoro modified nucleosides, 2'-deoxy modified nucleosides, locked nucleosides, unlocked nucleosides, nucleosides with restricted conformation, constrained ethyl nucleosides, abasic nucleosides, 2'-amino modified nucleosides, 2'-O-allyl modified nucleosides, 2'-C-alkyl modified nucleosides, 2'-hydroxy modified nucleosides, 2'-methoxyethyl modified nucleosides, 2'-O-alkyl modified nucleosides, morpholino nucleosides, phosphoramidates, nucleosides containing unnatural bases, tetrahydropyran modified nucleosides, 1,5-anhydrohexitol modified nucleosides, cyclohexenyl modified nucleosides, nucleosides containing phosphorothioate groups, nucleosides containing methylphosphonate groups, nucleosides containing 5'-phosphate, and nucleosides containing 5'-phosphate mimics. In another embodiment, the modified nucleoside comprises a short sequence of 3'-terminal deoxy-thymidine nucleoside (dT).

[0212] The modification of 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 of the nucleoside is a 2'O-methyl ("2-Me") or 2'-fluoro modification.

[0213] One preferred modification is a modification selected, optionally, from a 2'-Me modification or a 2'-F modification at the 2'-OH group of the ribose sugar.

[0214] Preferred nucleic acids contain one or more nucleosides in the first strand and / or the second strand, which are modified as follows to form modified nucleosides.

[0215] The modification is a modification selected optionally from a 2'-Me modification or a 2'-F modification at the 2'-OH group of the ribose sugar, nucleic acid.

[0216] The first strand is a nucleic acid containing a 2'-F modification at any one of positions 2, 6, 14, or any combination thereof, counted from position 1 of the first strand.

[0217] The second strand is a nucleic acid containing a 2'-F modification at any one of positions 7, 9, 11, or any combination thereof, counted from position 1 of the second strand.

[0218] The second strand is a nucleic acid containing a 2'-F modification at positions 7 and / or 9 and / or 11 and / or 13, counted from position 1 of the second strand.

[0219] The second strand is a nucleic acid containing 2'-F modifications at positions 7, 9, and 11, counted from position 1 of the second strand.

[0220] The first strand and the second strand each contain 2'-Me modifications and 2'-F modifications, nucleic acid.

[0221] A nucleic acid containing at least one thermolabilizing modification, preferably at one or more of positions 1 to 9 of the first strand, counted from position 1 of the first strand, and / or at one or more of the positions of 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, nucleic acid.

[0222] A nucleic acid containing 3 or more 2'-F modifications at positions 7 to 13 of the second strand, counted from position 1 of the second strand, for example, 4, 5, 6, or 7 2'-F modifications at positions 7 to 13 of the second strand.

[0223] The second strand is a nucleic acid containing at least 3, for example, 4, 5, or 6 2'-Me modifications at positions 1 to 6 of the second strand, counted from the 1st position of the second strand.

[0224] The first strand is preferably a nucleic acid containing at least 5 consecutive 2'-Me modifications in the 3'-terminal region, including the terminal nucleoside of the 3'-terminal region, or within at least 1 or 2 nucleosides from the terminal nucleoside of the 3'-terminal region.

[0225] The first strand is preferably a nucleic acid containing 7 consecutive 2'-Me modifications in the 3'-terminal region, including the terminal nucleoside of the 3'-terminal region.

[0226] A nucleic acid containing at least one heat destabilizing modification at the 7th position of the first strand, counted from the 1st position of the first strand.

[0227] A nucleic acid which is an siRNA oligonucleoside, wherein the siRNA oligonucleoside contains at least 3 2'-F modifications at positions 6 to 12 of the second strand, counted from the 1st position of the second strand.

[0228] A nucleic acid which is an siRNA oligonucleoside, wherein the second strand contains at least 3 2'-Me modifications at positions 1 to 6 of the second strand, counted from the 1st position of the second strand.

[0229] A nucleic acid that is an siRNA oligonucleoside, wherein each of the first strand and the second strand contains an alternating modification pattern, preferably a perfect alternating modification pattern, along the entire length of each of the first strand and the second strand, and the nucleosides of the first strand are modified by (i) 2’Me modification of the odd-numbered nucleosides counted from the 1st position of the first strand, and (ii) 2’F modification of the even-numbered nucleosides counted from the 1st position of the first strand, and the nucleosides of the second strand are modified by (i) 2’F modification of the odd-numbered nucleosides counted from the 1st position of the second strand, and (ii) 2’Me modification of the even-numbered nucleosides counted from the 1st position of the second strand. Typically, such a perfect alternating modification pattern is present in blunt-ended oligonucleosides, and each of the first strand and the second strand is 19 or 23 nucleosides in length.

[0230] The 1st position of the first strand or the second strand is closest to the end of the nucleic acid (any abasic nucleosides are ignored), and based on the bond between the sugar moieties of the backbone, when read in the direction away from that end of the molecule, it is the nucleoside joined to the adjacent nucleoside (the 2nd position) via an internal bond from 3’ to 5’.

[0231] Thus, it can be understood that the "1st position of the sense strand" is the 5’-most nucleoside of the conventional 5’ end of the sense strand (excluding abasic nucleosides). Typically, this 1st position nucleoside of the sense strand will be equivalent to the 5’ nucleoside of the selected target nucleic acid sequence. More generally, the sense strand will have nucleosides equivalent to those of the target nucleic acid sequence starting from this 1st position of the sense strand, although acceptable mismatches between sequences are also possible.

[0232] As used herein, the "1st position of the antisense strand" is the 5’-most nucleoside of the conventional 5’ end of the antisense strand (excluding abasic nucleosides). As described above, there will be a region of complementarity between the sense strand and the antisense strand, and thus the antisense strand will also have a region of complementarity to the target nucleic acid sequence referred to above.

[0233] In certain embodiments, a nucleic acid, such as an RNAi agent, further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage. For example, the phosphorothioate or methylphosphonate internucleoside linkage may be present at the 3' end or terminal region of one strand, i.e., the sense or antisense strand, or at the termini of both strands, the sense and antisense strands.

[0234] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkage may be present at the 5' end or terminal region of one strand, i.e., the sense or antisense strand, or at the termini of both strands, the sense and antisense strands.

[0235] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkage may be present at both the 5' and 3' ends 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.

[0236] Any of the nucleic acids may optionally 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.

[0237] At least one of the oligoribonucleoside strands preferably contains at least two consecutive phosphorothioate modifications in the last three nucleosides of the oligonucleoside.

[0238] Accordingly, the present invention also relates to a nucleic acid as disclosed herein, which comprises phosphorothioate internucleoside linkages between at least two or three consecutive positions, such as the 5' and / or 3' terminal regions and / or near-terminal regions of the second strand, wherein the near-terminal region preferably abuts the terminal region where the one or more abasic nucleosides of the second strand are located.

[0239] The nucleic acids disclosed herein each contain phosphorothioate internucleoside linkages between at least two or three consecutive positions in the 5' and / or 3' terminal regions of the first strand, and preferably, the terminal positions in the 5' and / or 3' terminal regions of the first strand are attached to their adjacent positions by phosphorothioate internucleoside linkages.

[0240] The nucleic acid strand may be an RNA containing phosphorothioate internucleoside linkages between three consecutive nucleosides that are contiguous to two terminal abasic nucleosides.

[0241] Preferred nucleic acids are double-stranded RNAs containing two adjacent abasic nucleosides at the 5' end of the second strand and a ligand moiety containing one or more GalNAc ligand moieties at the 3' end on the opposite side of the second strand. More preferably, the same nucleic acid may further contain phosphorothioate linkages between the nucleotides at positions 3-4 and 4-5 of the second strand when read from position 1 of the second strand. Even more preferably, the same nucleic acid may further contain 2'-F modifications at positions 7, 9, and 11 of the second strand.

[0242] The following structure:

[0243]

Chemical formula

[0244] The following structure

[0245]

Chemical formula

[0246] The modified nucleoside of the second strand has a modification pattern (5'-3') by any one of the following: Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me A nucleic acid comprising.

[0247] The modified nucleoside of the second strand has a modification pattern (5'-3') by any one of the following: Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, comprising a nucleic acid wherein (s) is a phosphorothioate internucleoside linkage.

[0248] The modified nucleosides of the second strand have a modification pattern (5'-3') by any one of the following: ia-ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or ia-ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, comprising wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in a 2-nucleoside overhang, a nucleic acid.

[0249] The modified nucleoside of the second strand has a modification pattern (5'-3') by any one of the following: ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, comprising wherein (s) is a phosphorothioate nucleoside internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in the overhang of two nucleosides, a nucleic acid.

[0250] The modified nucleoside has the following modification pattern: Modified pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 6: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-Me, nucleic acid containing any one of them.

[0251] The modified nucleoside has the following modified patterns: Modified pattern 1: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 2: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 3: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 4: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 5: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 6: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, containing any one of them, In the formula, (s) is a phosphorothioate internucleoside linkage, nucleic acid.

[0252] The modified nucleoside has the following modified pattern: Modified pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, first strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 6: Containing any one of the second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, first strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, Wherein (s) is a phosphorothioate nucleoside internucleoside linkage, a nucleic acid.

[0253] The modified nucleoside has the following modified pattern: Modified pattern 1: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 2: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me or modified pattern 3: second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me or modified pattern 4: second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me or modified pattern 5: second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me or modified pattern 6: second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, including any one of wherein ia represents an inverted abasic nucleoside, a nucleic acid.

[0254] The modified nucleoside has the following modified pattern: Modified pattern 1: second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 6: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, - First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, including any one of them, In the formula, ia represents an inverted abasic nucleoside. When the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is present in the overhang of two nucleosides, a nucleic acid.

[0255] The modified nucleoside has the following modification patterns: Modification pattern 1: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 2: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 3: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 4: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 5: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 6: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, containing any one of them, wherein, (s) is a phosphorothioate nucleoside internucleoside linkage, and ia represents an inverted abasic nucleoside, a nucleic acid.

[0256] The modified nucleoside has the following modified patterns: Modified pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, first strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 6: Containing any one of the following: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Wherein, (s) is a phosphorothioate nucleoside internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is present in a 2-nucleoside overhang, nucleic acid.

[0257] A nucleic acid, wherein the first strand contains 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 does not consist of 4 2’F modifications nor 6 2’F modifications.

[0258] A nucleic acid, wherein the first strand contains a 2’ sugar modification pattern, the modification is selected from at least 2’Me and 2’F sugar modifications, and the total number of 2’F sugar modifications in the first strand consists of 3, 5, or 7 2’F modifications.

[0259] A nucleic acid in which the first strand contains a 2'-sugar modification pattern, said 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 three 2'-F modifications.

[0260] A nucleic acid in which the first strand contains a 2'-sugar modification pattern, said 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 five 2'-F modifications.

[0261] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 wherein X2, X3, and X4 are selected from 2'-Me and 2'-F sugar modifications, provided that at least one of X2, X3, and X4 is a 2'-F sugar modification and the other two sugar modifications are 2'-Me sugar modifications.

[0262] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 wherein X2 is a 2'-F sugar modification and X3 and X4 are 2'-Me sugar modifications.

[0263] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 wherein X3 is a 2'-F sugar modification and X2 and X4 are 2'-Me sugar modifications.

[0264] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 wherein In the formula, X4 is a 2′-F sugar modification, and X2 and X3 are 2′-Me sugar modifications, nucleic acid.

[0265] The first strand contains a 2′-sugar modification pattern, the modification is selected from at least 2′-Me and 2′-F sugar modifications, and the total number of 2′-F sugar modifications of the first strand consists of 7 2′-F modifications, nucleic acid.

[0266] The first strand has the following 2′-sugar modification pattern (5′-3′): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 including In the formula, X2, X3, and X4 are selected from 2′-Me and 2′-F sugar modifications, provided that at least one of X2, X3, and X4 is a 2′-F sugar modification, and the other two sugar modifications are 2′-Me sugar modifications, nucleic acid.

[0267] The first strand has the following 2′-sugar modification pattern (5′-3′): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 including In the formula, X2 is a 2′-F sugar modification, and X3 and X4 are 2′-Me sugar modifications, nucleic acid.

[0268] The first strand has the following 2′-sugar modification pattern (5′-3′): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 including In the formula, X3 is a 2′-F sugar modification, and X2 and X4 are 2′-Me sugar modifications, nucleic acid.

[0269] The first strand has the following 2′-sugar modification pattern (5′-3′): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 including In the formula, X4 is a 2′-F sugar modification, and X2 and X3 are 2′-Me sugar modifications, nucleic acid.

[0270] The first strand has the following 2′-sugar modification pattern (5′-3′): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 including In the formula, X1 is a heat destabilizing modification, nucleic acid.

[0271] The first strand has the following 2′-sugar modification pattern (5′-3′): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 including In the formula, X1 is a heat destabilizing modification, nucleic acid.

[0272] The second strand has the following 2′-sugar modification pattern (5′-3′): (Me)8-(F)3-(Me) 10 including nucleic acid.

[0273] The second strand has the following 2′-sugar modification pattern (5′-3′): (Me)8-(F)3-(Me) 10 , including The first strand includes 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 neither four 2′-F modifications nor six 2′-F modifications, nucleic acid.

[0274] The second strand has the following 2′-sugar modification pattern (5′-3′): (Me)8-(F)3-(Me) 10 including The first strand includes a 2′-sugar modification pattern, the modification is selected from at least 2′-Me and 2′-F sugar modifications, and the total number of 2′-F sugar modifications in the first strand consists of three, five, or seven 2′-F modifications, nucleic acid.

[0275] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, wherein X1 is a heat destabilizing modification, nucleic acid.

[0276] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): (Me-F)3-(Me)7-F-Me-F-(Me)7 comprising, nucleic acid.

[0277] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 , comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5 A nucleic acid comprising

[0278] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 , comprising The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3 A nucleic acid comprising

[0279] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 , comprising The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7, wherein X1 is a thermally destabilizing modification, a nucleic acid.

[0280] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand having the following 2'-sugar modification pattern (5'-3'): (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6 a nucleic acid.

[0281] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand having the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5 a nucleic acid.

[0282] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand having the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3 a nucleic acid.

[0283] The second strand has the following 2'-sugar and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising a nucleic acid, wherein ia represents an inverted abasic nucleoside.

[0284] The second strand has the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The first strand comprises 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 does not consist of 4 2'-F modifications nor 6 2'-F modifications, a nucleic acid.

[0285] The second strand has the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The first strand comprises 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.

[0286] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, wherein X1 is a thermolabile modification, a nucleic acid.

[0287] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): (Me-F)3-(Me)7-F-Me-F-(Me)7 A nucleic acid comprising the above.

[0288] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5 A nucleic acid comprising the above.

[0289] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand are the following 2'-sugar modification patterns (5'-3'): Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3 A nucleic acid comprising.

[0290] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand are the following 2'-sugar modification and abasic modification patterns (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand are the following 2'-sugar modification patterns (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7, comprising, wherein X1 is a thermolabile modification, a nucleic acid.

[0291] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand are the following 2'-sugar modification and abasic modification patterns (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand are the following 2'-sugar modification patterns (5'-3'): (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6 A nucleic acid comprising.

[0292] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5 A nucleic acid comprising the same.

[0293] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3 A nucleic acid comprising the same.

[0294] A nucleic acid, wherein the second strand has the following 2'-sugar modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 comprising wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage.

[0295] The second strand has the following 2'-sugar modification pattern (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, The first strand contains a 2'-sugar modification pattern, said 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 neither four 2'-F modifications nor six 2'-F modifications, nucleic acid.

[0296] The second strand has the following 2'-sugar modification pattern (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, The first strand contains a 2'-sugar modification pattern, said 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 three, five, or seven 2'-F modifications, nucleic acid.

[0297] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-X1-(Me)7-F-Me-F-(Me)5(s)Me(s)Me, wherein X1 is a thermolabile modification, nucleic acid.

[0298] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)Me-F-Me-F-(Me)7-F-Me-F-(Me)5(s)Me(s)Me A nucleic acid comprising the same.

[0299] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-(Me)7-(F-Me)2-F-(Me)3(s)Me(s)Me A nucleic acid comprising the same.

[0300] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-(Me)7-F-Me-F-(Me)3-F-Me(s)Me(s)Me A nucleic acid comprising the same.

[0301] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, The nucleosides of the first strand have the following 2'-sugar modification pattern (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 thermolabile modification, A nucleic acid.

[0302] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)Me-F-Me-F-Me-(F)2-(Me)4-(F-Me)2-(Me)4(s)Me(s)Me A nucleic acid comprising the same.

[0303] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)3(s)Me(s)Me A nucleic acid comprising the same.

[0304] 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 strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (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, the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-Me(s)Me(s)Me A nucleic acid comprising the above.

[0305] 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 thermolabile 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-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me ; Or modification pattern 3: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me ; Or modification pattern 4: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-Me ; Or modification pattern 5: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, where X1 is a thermolabile modification; Or modification pattern 6: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me ; Or modification pattern 7: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-F-F-Me- Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me ; or modification pattern 8: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-Me

[0306] 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, where X1 is a heat destabilizing modification; or modification pattern 2: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me ; or modification pattern 3: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me; or modified pattern 4: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me ; or modified pattern 5: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where X1 is a thermally labile modification; or modified pattern 6: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me ; or modified pattern 7: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-F-F-Me - Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me ; or modified pattern 8: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me; wherein (s) represents a phosphorothioate internucleoside linkage.

[0307] Conjugation of Nucleic Acids and Ligands Another modification of the nucleic acids of the present invention, such as RNA, such as siRNA, involves conjugating the nucleic acid, such as siRNA, to one or more ligand moieties to enhance, for example, the activity, cellular distribution, or cellular uptake into cells of the nucleic acid, such as siRNA.

[0308] In some embodiments, the described ligand moieties may be attached to a nucleic acid, such as an siRNA oligonucleoside, via a linker that 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, attaches two parts of a compound by a covalent bond.

[0309] The ligand can be attached to the 3’ or 5’ end of the sense strand.

[0310] The ligand is preferably conjugated to the 3’ end of the sense strand of a nucleic acid, such as an siRNA agent.

[0311] Accordingly, in a further aspect, the present invention relates to a conjugate for inhibiting the expression of a target, such as a target gene, in a cell, said conjugate comprising a nucleic acid moiety and one or more ligand moieties, wherein said nucleic acid moiety comprises a nucleic acid disclosed herein.

[0312] 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, and said ligand moieties are typically present in the terminal region of the second strand, preferably in its 3' terminal region.

[0313] In certain embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative attached to the nucleic acid, e.g., dsiRNA, via a linker.

[0314] Accordingly, the present invention relates to conjugates comprising a ligand moiety that i) one or more GalNAc ligands, and / or ii) one or more GalNAc ligand derivatives, and / or iii) one or more GalNAc ligands conjugated to said nucleic acid via a linker is included.

[0315] The GalNAc ligand may be conjugated directly or indirectly to the 5' or 3' terminal region of the second strand of the nucleic acid, preferably to its 3' terminal region.

[0316] GalNAc ligands are well known in the art and are described, inter alia, in European Patent No. 3775207.

[0317] In some embodiments, the ligand moiety comprises one or more ligands.

[0318] In some embodiments, the ligand moiety comprises one or more carbohydrate ligands.

[0319] In some embodiments, the one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and / or polysaccharides.

[0320] In some embodiments, the one or more carbohydrates include one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0321] In some embodiments, the one or more carbohydrates include one or more N-acetyl-galactosamine moieties.

[0322] In some embodiments, the compounds described anywhere herein include two or three N-acetylgalactosamine moieties.

[0323] In some embodiments, the one or more ligands are attached in a linear configuration or a branched configuration, for example, each configuration is attached to a branching point of the entire linker.

[0324] Exemplary linear configurations and exemplary branched configurations are shown in FIGS. 1a and 1b.

[0325] In FIG. 1a (linear), (a) and / or (b) can typically represent a connecting bond or a connecting group such as a phosphate group or a phosphorothioate group.

[0326] In FIG. 1b (branched), in some embodiments, the one or more ligands are attached as a bifurcated or trifurcated branched configuration. Typically, a trifurcated branched configuration such as an N-acetylgalactosamine trifurcated branched configuration may be preferred.

[0327] Linker Exemplary compounds of the present invention include a "linker portion" that is part of all "linkers", such as those illustrated in formula (I).

[0328] [Chemical formula] Wherein, R1, independently at each occurrence, is selected from the group consisting of hydrogen, methyl, and ethyl, R2 is hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo, and nitro, X1 and X2, independently at each occurrence, are 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 at the same time, (ii) s, t, and v cannot all be 0 at the same time, Z is an oligonucleoside moiety.

[0329] As will be further understood in the art, exemplary compounds of the invention include all linkers positioned between the oligonucleoside moiety and the ligand moiety of such compounds. The all linkers thereby "link" the oligonucleoside moiety and the ligand moiety to each other.

[0330] A linker is often conceptually envisioned to include one or more linker building blocks. For example, there is a linker portion illustrated as the "linker portion" presented in formula (I), which is disposed adjacent to the ligand portion and typically attaches the ligand portion to the oligonucleoside portion directly or indirectly via a branching point. The linker portion illustrated in formula (I) is often also referred to as the "ligand arm or group of arms" of the linker. There may further be an additional linker portion between the oligonucleoside portion and the branching point, but it does not always exist. The additional linker portion is often referred to as the "tether portion" of the linker that "tethers" the oligonucleoside portion to the remainder of the conjugate compound. Such "ligand arms" and / or "linker portions" and / or "tether portions" can be envisioned by referring to the linear and / or branched chain configurations shown above.

[0331] As can be understood from the claims and the remainder of this patent specification, the scope of the present invention extends to linear or branched chain configurations and there is no limitation on the number of individual ligands that may be present. Further, the reader will also recognize that there are numerous structures that can be used as linker portions based on the state of the art and the expertise of oligonucleoside chemists.

[0332] The remainder of the linker (other than the linker portion) shown in the claims and the remainder of the patent specification is represented by its chemical composition in formula (I) which the inventors consider to be particularly unique to the present invention. However, more generally, such chemical compositions can be described as the "tether portion" as described above, and the "tether portion" is the portion of the linker that includes the group of atoms Z, i.e., between the oligonucleoside portion and the linker portion, as illustrated in formula (I).

[0333] The tether portion of formula I Regarding formula (I), the "tether portion" includes the atomic group between Z, i.e., the oligonucleoside portion, and the linker portion.

[0334] In some embodiments, R1 is hydrogen at each occurrence. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl.

[0335] In some embodiments, R2 is hydroxy. In some embodiments, R2 is halo. In some embodiments, R2 is fluoro. In some embodiments, R2 is chloro. In some embodiments, R2 is bromo. In some embodiments, R2 is iodo. In some embodiments, R2 is nitro.

[0336] In some embodiments, X1 is methylene. In some embodiments, X1 is oxygen. In some embodiments, X1 is sulfur.

[0337] In some embodiments, X2 is methylene. In some embodiments, X2 is oxygen. In some embodiments, X2 is sulfur.

[0338] In some embodiments, m = 3.

[0339] In some embodiments, n = 6.

[0340] In some embodiments, X1 is oxygen and X2 is methylene. In some embodiments, both X1 and X2 are methylene.

[0341] In some embodiments, q = 1, r = 2, s = 1, t = 1, v = 1. In some embodiments, q = 1, r = 3, s = 1, t = 1, v = 1.

[0342] In some embodiments, R1 is hydrogen at each occurrence, n = 6, m = 3, R2 is fluoro, X2 is methylene, v = 1, t = 1, s = 1, X1 is methylene, q = 1, and r = 2.

[0343] Thus, in some embodiments, exemplary compounds of the invention include the following structure.

[0344]

Chemical formula

[0345] In some embodiments, R1 is hydrogen at each occurrence, n = 6, m = 3, R2 is fluoro, X2 is methylene, v = 1, t = 1, s = 1, X1 is oxygen, q = 1, and r = 2.

[0346] Thus, in some embodiments, exemplary compounds of the invention include the following structure.

[0347]

Chemical formula

[0348] Alternative tether moiety During the synthesis of the compounds of the invention, alternative tether moiety structures may occur. In some embodiments, the alternative tether moiety has a change in one or more atoms of the tether moiety of the entire linker as compared to the tether moiety described anywhere in this specification.

[0349] In some embodiments, the alternative tether moiety is a compound of formula (I) described anywhere in this specification, wherein R2 is hydroxy.

[0350] In some embodiments, R1 is hydrogen at each occurrence, n = 6, m = 3, R2 is hydroxy, X2 is methylene, v = 1, t = 1, s = 1, X1 is methylene, q = 1, and r = 2.

[0351] Thus, in some embodiments, the compounds of the invention include the following structure.

[0352]

Chem.

[0353] In some embodiments, R1 is hydrogen at each occurrence, n = 6, m = 3, R2 is hydroxy, X2 is methylene, v = 1, t = 1, s = 1, X1 is oxygen, q = 1, and r = 2.

[0354] Thus, in some embodiments, the compounds of the present invention include the following structure.

[0355]

Chem.

[0356] Linker moiety With respect to formula (I), the "linker moiety" illustrated in formula (I) includes a group of atoms located between the tether moiety described anywhere in this specification and the ligand moiety described anywhere in this specification.

[0357] In some embodiments, it is illustrated in formula (I) described anywhere in this specification.

[0358]

Chem.

[0359]

Chem.

[0360] [Chemical formula] 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 an integer of 1 to 6, or

[0361] [Chemical formula] 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.

[0362] In some embodiments, the portion illustrated in formula (I):

[0363] [Chemical formula] is formula (VIa),

[0364] [Chemical formula] In the formula, A I is hydrogen or a suitable hydroxy protecting group, a is 3, b is an integer of 3.

[0365] In some embodiments, the portion illustrated in formula (I) described anywhere in this specification:

[0366] [Chemical formula] is formula (VII),

[0367] [Chem.] In the formula, A I is hydrogen, a is an integer of 2 or 3, preferably 3.

[0368] Another exemplary compound of the present invention includes a "linker portion" that is part of all "linkers" illustrated in formula (I * ), and

[0369] [Chem.] In the formula, r and s are independently integers selected from 1 to 16, Z is an oligonucleoside moiety.

[0370] As will be further understood in the art, exemplary compounds of the present invention include all linkers located between the oligonucleoside moiety and the ligand moiety of such compounds. The all linkers thereby "link" the oligonucleoside moiety and the ligand moiety to each other.

[0371] All linkers are often conceptually envisioned to include one or more linker building blocks. For example, there is a linker portion illustrated as the "linker portion" presented in formula (I * ), and this linker portion is disposed adjacent to the ligand moiety and typically attaches the ligand moiety to the oligonucleoside moiety directly or indirectly via a branching point. Formula (I *) The linker portion illustrated in [0] is often also referred to as the "ligand arm or group of arms" of the entire linker. There may be an additional linker portion between the oligonucleoside portion and the branching point, but it does not always exist. The additional linker portion is often referred to as the "tether portion" of the entire linker, which "tethers" the oligonucleoside portion to the rest of the conjugate compound. Such "ligand arms" and / or "linker portions" and / or "tether portions" can be envisioned by referring to the linear and / or branched chain configurations shown above.

[0372] As can be understood from the claims and the remainder of this patent specification, the scope of the present invention extends to linear or branched chain configurations and is not limited by the number of individual ligands that may be present. Furthermore, the reader will also recognize that there are numerous structures that can be used as linker portions based on the state of the art and the expertise of oligonucleoside chemists.

[0373] The remainder of the entire linker (other than the linker portion) shown in the claims and the remainder of the patent specification is represented by its chemical composition in formula (I), which the inventors consider to be particularly unique to the present invention. However, more generally speaking, such chemical compositions can be described as the "tether portion" as described above, and the "tether portion" is the portion of the entire linker that includes the group of atoms between Z, i.e., the oligonucleoside portion and the linker portion, as illustrated in formula (I).

[0374] Tether portion Formula (I * ) With respect to, the "tether portion" includes the group of atoms between Z, i.e., the oligonucleoside portion and the linker portion.

[0375] In some embodiments, s is an integer selected from 4 to 12. In some embodiments, s is 6.

[0376] In some embodiments, r is an integer selected from 4 to 14. In some embodiments, r is 6. In some embodiments, r is 12.

[0377] In some embodiments, r is 12 and s is 6.

[0378] Accordingly, in some embodiments, exemplary compounds of the present invention include the following structure.

[0379]

Chemical formula

[0380] In some embodiments, r is 6 and s is 6.

[0381] Accordingly, in some embodiments, exemplary compounds of the present invention include the following structure.

[0382]

Chemical formula

[0383] Linker moiety With respect to formula (I * ), the "linker moiety" illustrated in formula (I) includes a group of atoms located between the tether moiety described anywhere in this specification and the ligand moiety described anywhere in this specification.

[0384] In some embodiments, the moiety illustrated in formula (I * ) described anywhere in this specification:

[0385]

Chemical formula

[0386]

Chem.

[0387]

Chem.

[0388]

Chem.

[0389] In some embodiments, the portion shown in formula (I):

[0390]

Chem.

[0391]

Chem.

[0392] In some embodiments, the moiety illustrated in formula (I) described anywhere in this specification:

[0393]

Chemical formula

[0394]

Chemical formula

[0395] In some embodiments, a = 2. In some embodiments, a = 3. In some embodiments, b = 3.

[0396] Vectors and cells In one aspect, the present invention provides a cell comprising a nucleic acid such as an inhibitory RNA [RNAi] described herein.

[0397] In one aspect, the present invention provides a cell comprising a vector described herein.

[0398] In one aspect, the present invention provides a vector comprising an oligonucleotide inhibitor, such as an iRNA, such as an siRNA.

[0399] Pharmaceutically acceptable compositions In one aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of a target gene, the composition comprising an inhibitor such as an oligomer such as a nucleic acid disclosed herein.

[0400] The pharmaceutically acceptable composition may contain excipients and / or carriers.

[0401] Some examples of substances that can serve as pharmaceutically acceptable carriers include the following: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch, potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth powder; (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, 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.

[0402] Typical pharmaceutical carriers include, but are not limited to, the following: 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.).

[0403] For formulating the composition of the present invention, pharmaceutically acceptable organic or inorganic excipients that do not cause adverse reactions with nucleic acids and are suitable for parenteral administration can also be used. 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, and polyvinylpyrrolidone, etc.

[0404] 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 bases. The solution may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not cause adverse reactions with nucleic acids and are suitable for parenteral administration can be used.

[0405] 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, such as an RNAi agent, is administered in a buffered solution. In such embodiments, the buffer solution may contain acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffer solution may be phosphate buffered saline (PBS).

[0406] Dosage The pharmaceutical composition of the present invention can be administered in a dosage sufficient to inhibit gene expression or modify the expression or function of a target such as lncRNA. Generally, when the composition contains a nucleic acid, a suitable dosage of the nucleic acid of the present invention, such as siRNA, is in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, and generally will be 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, such as siRNA, will be in the range of about 0.1 mg / kg to about 5.0 mg / kg, such as about 0.3 mg / kg to about 3.0 mg / kg.

[0407] A repeated dosage regimen may include administering a therapeutic amount of a nucleic acid, such as siRNA, periodically, for example, once a day or once a year. In certain embodiments, the nucleic acid, such as siRNA, is administered about once a month to once every three months (i.e., once every three months).

[0408] 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 siRNA 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 once a quarter (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 about once every quarter (i.e., about once every three months).

[0409] After the initial treatment regimen, treatment may be administered less frequently. For example, after administration once a week or once every two weeks over a three-month period, administration may be repeated once a month, once every six months, or once a year or longer.

[0410] The pharmaceutical composition may be administered once a day, or alternatively, as two, three, or more divided doses at appropriate intervals throughout the day, or even using delivery by sustained infusion or controlled release formulations. In that case, the nucleic acid, such as siRNA, contained in each divided dose will have to be proportionally less in order to achieve the total daily dosage. Dosage units can also be formulated to be delivered over several days, for example, using conventional sustained release formulations that provide 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 a corresponding multiple of the daily dose.

[0411] In other embodiments, a single dose of the pharmaceutical composition may have long-term persistence, such that subsequent doses are administered at intervals within 3, 4, or 5 days, or at intervals within 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 once every two months. In certain embodiments, the siRNA is administered about once a month to about once every quarter (i.e., about once every three months), or even once every six months or once every twelve months.

[0412] The estimation of the effective dosage and in vivo half-life of the individual nucleic acids, such as siRNA, encompassed by the present invention can be carried out using conventional methodologies or based on in vivo tests using appropriate animal models known in the art.

[0413] The pharmaceutical composition of the present invention can be administered in a number of ways depending on whether local or systemic treatment is desired and depending on the site to be treated. Administration can be local (e.g., by transdermal patch), pulmonary, such as by inhalation or insufflation of a powder or aerosol, including by nebulizer, intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subcutaneous, e.g., by an implantable device, or intracranial, e.g., intrasubstantial, 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.

[0414] In one embodiment, the nucleic acid, such as the siRNA agent, is administered subcutaneously to a subject.

[0415] Inhibitors, such as nucleic acids, such as siRNA, can be delivered to target specific tissues (e.g., specific hepatocytes).

[0416] Methods for inhibiting gene expression, or methods for inhibiting target expression or function The present invention also provides a method for inhibiting gene expression in a cell, as well as a method for inhibiting the expression and / or function of other target molecules such as lncRNA. Such methods include contacting the cell with an effective amount of a nucleic acid of the present invention for inhibiting gene expression in the cell, such as an siRNA agent, such as a double-stranded siRNA agent, thereby inhibiting gene expression in the cell. In a preferred embodiment, the gene encodes an enzyme involved in post-translational glycosylation. In a more preferred embodiment, the gene is B4GALT1.

[0417] Contact of the cell with an inhibitor, such as a nucleic acid, such as siRNA, such as a double-stranded siRNA agent, can be carried out in vitro or in vivo. Contacting the cell with an inhibitor nucleic acid, such as siRNA, in vivo includes contacting a cell or cell population within a subject, such as a human subject, with the nucleic acid, such as siRNA. Combinations of in vitro and in vivo methods for contacting the cell are also possible. As discussed above, the contact with the cell may be direct or indirect. Further, the contact with the cell can be achieved by a target-directed ligand moiety comprising any ligand moiety described herein or known in the art. In a preferred embodiment, the target-directed 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.

[0418] The term "inhibit" as used herein is used interchangeably with "reduce", "silence", "down-regulate", "suppress", and other similar terms and includes any level of inhibition.

[0419] In some embodiments of the methods of the invention, the expression or activity of a gene or inhibitory target, such as an lncRNA, is preferably inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% as determined by qPCR as described herein and / or when siRNA is introduced into target cells by transfection, or inhibited to a level below the detection level of the assay. In certain embodiments, such methods include clinically relevant inhibition of the expression of a target gene, such as demonstrated by clinically relevant outcomes after treating a subject with an agent that reduces the expression and / or activity of the gene.

[0420] In some embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the B4GALT1 gene with an IC50 value lower 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 as described herein, more preferably by reverse transcriptase (RT)-qPCR.

[0421] In preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the B4GALT1 gene with an IC50 value lower than 2500 pM. In more preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the B4GALT1 gene with an IC50 value lower than 1000 pM. In even more preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the B4GALT1 gene with an IC50 value lower than 500 pM. In most preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the B4GALT1 gene with an IC50 value lower than 100 pM.

[0422] Inhibition of B4GALT1 gene expression can be quantified using the following method.

[0423] Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB cell bank) may be maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere. Next, siRNA duplexes targeting B4GALT1 mRNA or negative control siRNA (siRNA control, sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 623), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 622)) may be transfected into the cells using 10×3-fold serial dilutions over a final duplex 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 may be incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells may be incubated at 37 °C / 5% CO2 for 24 hours before purifying total RNA using the RNeasy 96 kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in a single experiment.

[0424] cDNA synthesis can be carried out using the FastQuant RT (with gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be carried out using the TaqMan Gene Expression Assay kit (ThermoFisher Scientific) with primers specific for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or ABI QuantStudio 7.

[0425] qPCR can be performed in duplicate on cDNA derived from each well to calculate the average cycle threshold (Ct). Relative B4GALT1 expression can be calculated from the average Ct values using the comparative Ct (ΔΔCt) method and normalized to GAPDH and to untreated cells. The maximum percent inhibition and IC50 value of B4GALT1 expression can be calculated using a four-parameter (variable slope) model using GraphPad Prism9.

[0426] Alternatively or in addition, the inhibitory ability of the nucleic acids of the invention can be quantified without prior transfection of the nucleic acids into the target cells.

[0427] Thus, in some embodiments, when cells are incubated with the nucleic acids of the invention, the nucleic acids of the invention preferably inhibit the expression of the B4GALT1 gene with an EC50 value lower than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, or 100 nM, as determined by qPCR, more preferably reverse transcriptase (RT)-qPCR, as described herein.

[0428] In preferred embodiments, when cells are incubated with the nucleic acids of the invention, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an EC50 value lower than 1000 nM. In more preferred embodiments, when cells are incubated with the nucleic acids of the invention, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an EC50 value lower than 500 nM. In even more preferred embodiments, when cells are incubated with the nucleic acids of the invention, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an EC50 value lower than 200 nM. In the most preferred embodiments, when cells are incubated with the nucleic acids of the invention, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an EC50 value lower than 100 nM.

[0429] Inhibition of B4GALT1 gene expression in the presence of free nucleic acids can be quantified using the following method.

[0430] Primary C57BL / 6 mouse hepatocytes (PMH) can be freshly isolated by two-step collagenase liver perfusion. The cells can be maintained in DMEM (Gibco - 11995 - 092) supplemented with FBS, penicillin / streptomycin, HEPES, and L-glutamine. The cells can be cultured at 37 °C in a 5% CO2 atmosphere in a humidified incubator. Within 2 hours after isolation, PMH may be seeded at a density of 36,000 cells / well in a normal 96-well tissue culture plate. Dose-response analysis in PMH can be performed by directly incubating the cells with a set of gymnotic free uptake at final GalNAc-siRNA concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM. In control wells, the cells can be incubated without GalNAc-siRNA. After 48 hours of culture, the cells can be harvested for RNA extraction. Total RNA can be extracted using the RNeasy kit according to the manufacturer's instructions (Qiagen, Shanghai, China). After reverse transcription, real-time quantitative PCR can be performed using ABI Prism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH. The expression of the target gene in each test sample can be determined by relative quantification using the comparative Ct (ΔΔCt) method. In this method, the Ct difference (ΔCt) between the target gene and the housekeeping gene is measured. The formulas are as follows: ΔCt = average Ct of B4GALT1 - average Ct of GAPDH, ΔΔCt = ΔCt (sample) - average ΔCt (untreated control), relative expression of target gene mRNA = 2 -ΔΔCt 。

[0431] Alternatively or in addition, inhibition of B4GALT1 gene expression can be characterized by a reduction in the average relative expression of the B4GALT1 gene.

[0432] In some embodiments, when cells are transfected with 0.1 nM of the nucleic acid of the present invention, the average relative expression of B4GALT1 is preferably measured by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein, and is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4.

[0433] In some embodiments, when cells are transfected with 5 nM of the nucleic acid of the present invention, the average relative expression of B4GALT1 is preferably measured by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein, and is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3.

[0434] The average relative expression of the B4GALT1 gene can be quantified using the following method.

[0435] Huh7 cells (a human hepatocyte-derived cell line, obtained from the JCRB cell bank) may be maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 °C in a 5% CO₂ atmosphere. siRNA duplex targeting B4GALT1 mRNA or negative control siRNA (siRNA control, sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 623), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 622)) may be transfected into the cells 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 may be incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells may be incubated at 37 °C / 5% CO₂ for 24 hours before purifying total RNA using the RNeasy 96 kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in two independent experiments.

[0436] cDNA synthesis can be carried out using the FastQuant RT (with gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed using the TaqMan Gene Expression Assay kit (ThermoFisher Scientific) with primers specific for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or ABI QuantStudio 7.

[0437] qPCR can be performed in duplicate on cDNA from each well to calculate the average Ct. Relative B4GALT1 expression can be calculated from the average Ct values using the comparative Ct (ΔΔCt) method and normalized to GAPDH and to untreated cells.

[0438] Inhibition of gene expression may be represented by a reduction in the amount of mRNA of the target gene of interest compared to a suitable control. Inhibition of target function may be represented by a reduction in the activity of the target compared to a suitable control.

[0439] In other embodiments, inhibition of gene or other target expression may be evaluated in terms of a reduction in gene expression, such as protein expression or a parameter functionally related to a signaling pathway.

[0440] Method for treating or preventing a disease associated with the expression of a target, such as the gene expression / function of LCNRNA The present invention also provides a method for reducing or inhibiting gene expression in a cell or for reducing the expression or function of a target using a nucleic acid of the present invention, such as siRNA, or a composition comprising a nucleic acid of the present invention, such as siRNA. Such methods include 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 mRNA transcript of the gene, thereby inhibiting gene expression in the cell. Reduction of target gene expression or function can be evaluated by any method known in the art. In a preferred embodiment, the gene encodes an enzyme involved in post-translational glycosylation. In a more preferred embodiment, the gene is B4GALT1.

[0441] In the method of the present invention, the cell may be contacted in vitro or in vivo, i.e., the cell may be present within a subject.

[0442] A cell suitable for treatment using the method of the present invention may be any cell that expresses the target gene or target of interest associated with the disease.

[0443] The in vivo method of the present invention comprises administering to a subject a composition comprising a nucleic acid of the present invention, such as siRNA, wherein the nucleic acid, such as siRNA, comprises a nucleoside sequence complementary to at least a part of the RNA transcript of a gene of a mammal to be treated, or complementary to another nucleic acid whose expression and / or function is associated with a disease.

[0444] The present invention further provides a method for treating a subject in need thereof. The treatment method of the present invention comprises administering to a subject, for example, a nucleic acid such as siRNA of the present invention, a nucleic acid such as siRNA targeting a gene, or a pharmaceutical composition comprising a nucleic acid targeting a gene, in a therapeutically effective amount to a subject who would benefit from a reduction or inhibition of gene expression and / or expression and / or function of a target.

[0445] The nucleic acid of the present invention, such as siRNA, may be administered as a "free" nucleic acid or "free" siRNA administered in the absence of a pharmaceutical composition. The naked nucleic acid may be present 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 osmotic pressure of the buffer solution can be adjusted to be suitable for administration to a subject.

[0446] Alternatively, the nucleic acid of the present invention, such as siRNA, may be administered as a pharmaceutical composition such as a dsiRNA liposome formulation.

[0447] In one embodiment, the method comprises administering the composition described herein such that the expression of the target gene decreases over, for example, 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 decreases for a long period of time, for example, at least about 2, 3, 4 days or longer, for example, about 1 week, 2 weeks, 3 weeks, or 4 weeks, or longer, for example, about 1 month, 2 months, or 3 months.

[0448] A therapeutically effective amount of a nucleic acid, such as, for example, about 0.01 mg / kg to about 200 mg / kg of siRNA, can be administered to the subject.

[0449] The nucleic acid, such as siRNA, can be administered by intravenous infusion over a period of time on a regular basis. In certain embodiments, after an initial treatment regimen, treatment may be administered at a lower frequency. Administration of siRNA can, for example, reduce the gene product level of a target gene in a patient's cells or tissues 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 used. In certain embodiments, administration results in clinical stabilization or preferably a clinically relevant reduction of at least one sign or symptom of the gene-related disorder.

[0450] Alternatively, the 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 the nucleic acid, such as siRNA, to the subject. The injections may be repeated over a period of time. Administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, treatment may be administered at a lower frequency. A repeated dose regimen may include administering a therapeutically effective amount of the nucleic acid regularly, for example, once a day to once a year. In certain embodiments, the nucleic acid is administered about once a month to about once every quarter (i.e., about once every three months).

[0451] In one aspect, the present invention can be applied to the compounds, methods, compositions, or uses of Proposition Numbers 1 to 101 below (references to any of the formulas of Propositions 1 to 101 refer only to the formulas defined within Propositions 1 to 101. Such formulas are reproduced in Figure 6).

[0452] 1. The following structure:

[0453]

Chemical formula

[0454] 2. The compound according to claim 1, wherein R1 is hydrogen in each occurrence.

[0455] 3. The compound according to claim 1, wherein R1 is methyl.

[0456] 4. The compound according to claim 1, wherein R1 is ethyl.

[0457] 5. The compound according to any one of claims 1 to 4, wherein R2 is hydroxy.

[0458] 6. The compound according to any one of claims 1 to 4, wherein R2 is halo.

[0459] 7. The compound according to claim 6, wherein R2 is fluoro.

[0460] 8. The compound according to claim 6, wherein R2 is chloro.

[0461] 9. The compound according to claim 6, wherein R2 is bromo.

[0462] 10. The compound according to claim 6, wherein R2 is iodo.

[0463] 11. The compound according to claim 6, wherein R2 is nitro.

[0464] 12. The compound according to any one of claims 1 to 11, wherein X1 is methylene.

[0465] 13. The compound according to any one of claims 1 to 11, wherein X1 is oxygen.

[0466] 14. The compound according to any one of claims 1 to 11, wherein X1 is sulfur.

[0467] 15. The compound according to any one of claims 1 to 14, wherein X2 is methylene.

[0468] 16. The compound according to any one of claims 1 to 15, wherein X2 is oxygen.

[0469] 17. The compound according to any one of claims 1 to 16, wherein X2 is sulfur.

[0470] 18. The compound according to any one of claims 1 to 17, wherein m = 3.

[0471] 19. The compound according to any one of claims 1 to 18, wherein n = 6.

[0472] 20. X1 is oxygen, X2 is methylene, and preferably, q = 1, r = 2, s = 1, t = 1, v = 1, the compound according to claims 13 and 15.

[0473] 21. Both X1 and X2 are methylene, preferably, q = 1, r = 3, s = 1, t = 1, v = 1, the compounds described in Propositions 12 and 15.

[0474] 22. Z is,

[0475]

Chemical formula

[0476] 23. The oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene, the compound described in Proposition 22.

[0477] 24. The RNA compound comprises an RNA duplex containing a first strand 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, and each of the first strand and the second strand has 5' and 3' ends, the compound described in Proposition 23.

[0478] 25. The RNA compound has an adjacent phosphate attached at the 5' end of its second strand, the compound described in Proposition 24.

[0479] 26. The RNA compound has an adjacent phosphate attached at the 3' end of its second strand, the compound described in Proposition 24.

[0480] 27. The compound of formula (II).

[0481] [Chemistry]

[0482] 28. The compound of formula (III).

[0483] [Chemistry]

[0484] 29. The oligonucleoside comprises an RNA duplex containing a first strand 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 strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand, and is the compound according to Proposition 27 or 28.

[0485] 30. A composition comprising the compound of formula (II) defined in Proposition 27 and the compound of formula (III) defined in Proposition 28, and optionally subordinate to Proposition 29.

[0486] 31. The composition according to Proposition 30, wherein the compound of formula (III) defined in Proposition 28 is present in an amount in the range of 10 to 15% by weight of the composition.

[0487] 32. The compound of formula (IV).

[0488] [Chemistry]

[0489] 33. The compound of formula (V).

[0490] [Chemistry]

[0491] 34. An oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, the first strand being at least partially complementary to an RNA sequence of a target gene, the second strand being at least partially complementary to the first strand, each of the first strand and the second strand having 5' and 3' termini, and the RNA duplex being attached to an adjacent phosphate at the 3' terminus of its second strand, the compound according to claim 32 or 33.

[0492] 35. A composition comprising the compound of formula (IV) defined in claim 32 and the compound of formula (V) defined in claim 33, optionally dependent on claim 34.

[0493] 36. The composition according to claim 35, wherein the compound of formula (V) defined in claim 33 is present in an amount in the range of 10 to 15% by weight of the composition.

[0494] 37. An oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position, the compound defined in any of claims 1 to 29 or 32 to 34.

[0495] 38. The modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy, the compound according to claim 37.

[0496] 39. An oligonucleoside further comprises one or more degradation protection moieties at one or more termini, the compound according to any of claims 1 to 29, 32 to 34, or 37 to 38.

[0497] 40. The one or more degradation protection moieties are not present at the termini of the oligonucleoside strand carrying the ligand moiety, and / or the one or more degradation protection moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleosides are present at the distal end of the strand carrying the ligand moiety, the compound according to claim 39.

[0498] 41. The ligand moiety shown in formula (I) of Proposition 1 is a compound according to any of Propositions 1 to 29, 32 to 34, or 37 to 40, comprising one or more ligands.

[0499] 42. The ligand moiety shown in formula (I) of Proposition 1 is a compound according to Proposition 41, comprising one or more carbohydrate ligands.

[0500] 43. The one or more carbohydrates in the compound according to Proposition 42 may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[0501] 44. The one or more carbohydrates in the compound according to Proposition 43 comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0502] 45. The one or more carbohydrates in the compound according to Proposition 44 comprise one or more N-acetyl-galactosamine moieties.

[0503] 46. The compound according to Proposition 45 comprises two or three N-acetylgalactosamine moieties.

[0504] 47. The one or more ligands in the compound according to any of Propositions 41 to 46 are attached in a linear or branched configuration.

[0505] 48. The one or more ligands in the compound according to Proposition 47 are attached as a bifurcated or trifurcated branched configuration.

[0506] 49. The moiety shown in formula (I) of Proposition 1:

[0507]

Chemical formula

[0508]

Chemical formula

[0509]

Chemical formula

[0510]

Chemical formula

[0511] 50. The said part illustrated in formula (I) of Proposition 1:

[0512]

Chemical formula

[0513] [Chemistry] wherein A I is hydrogen, a is an integer of 2 or 3, the compounds described in Propositions 46 to 48.

[0514] 51. The compound according to Proposition 49 or 50, wherein a = 2.

[0515] 52. The compound according to Proposition 49 or 50, wherein a = 3.

[0516] 53. The compound according to Proposition 49, wherein b = 3.

[0517] 54. The compound of formula (VIII).

[0518] [Chemistry]

[0519] 55. The compound of formula (IX).

[0520] [Chemistry]

[0521] 56. The oligonucleoside contains an RNA duplex comprising a first strand 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 strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand. The compound according to Proposition 54 or 55.

[0522] 57. A composition comprising the compound of formula (VIII) defined in Proposition 54 and the compound of formula (IX) defined in Proposition 55, optionally dependent on Proposition 56.

[0523] 58. The composition according to Proposition 57, wherein the compound of formula (IX) defined in Proposition 55 is present in an amount in the range of 10 to 15% by weight of the composition.

[0524] 59. A compound of formula (X).

[0525] [Chemical formula]

[0526] 60. A compound of formula (XI).

[0527] [Chemical formula]

[0528] 61. The oligonucleoside comprises an RNA duplex comprising a first strand 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 strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand, and is the compound according to Proposition 59 or 60.

[0529] 62. A composition comprising the compound of formula (X) defined in Proposition 59 and the compound of formula (XI) defined in Proposition 60, and optionally dependent on Proposition 61.

[0530] 63. The composition according to Proposition 62, wherein the compound of formula (XI) defined in Proposition 60 is present in an amount in the range of 10 to 15% by weight of the composition.

[0531] 64. The compound defined in any of Propositions 54 to 63, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0532] 65. The modification is a compound according to claim 64 selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0533] 66. The oligonucleoside is a compound according to any one of claims 54 to 65 further comprising one or more deprotecting moieties at one or more termini.

[0534] 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, the inverted abasic nucleosides being present at the distal end of the chain carrying the ligand moiety as shown in any one of formulas (VIII), (IX), (X), or (XI) of claims 54, 55, 59, or 60, a compound according to claim 66.

[0535] 68. A method for preparing a compound according to any one of claims 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of claims 30, 31, 35, 36, 57, 58, 62, 63, the compounds of formulas (XII) and (XIII):

[0536]

Chemical formula

[0537] 69. The compound of formula (XII) is the compounds of formulas (XIV) and (XV):

[0538]

Chemical formula

[0539] 70. The compound of formula (XII) is of formula (XIIa),

[0540]

Chemical formula

[0541]

Chem.

[0542] 71. The compound of formula (XII) is of formula (XIIb),

[0543]

Chem.

[0544]

Chem.

[0545] 72. The compound of formula (XII) is of formula (XIIc),

[0546]

Chem.

[0547]

Chem.

[0548] 73. The compound of formula (XII) is of formula (XIId),

[0549]

Chem.

[0550]

Chem.

[0551] 74. The compound of formula (XIIIa) is of formula (XIIIb):

[0552] [Chemical formula] The method according to any one of claims 70 to 73, wherein it is as follows.

[0553] 75. The compound of formula (XIV) is either of formula (XIVa) or formula (XIVb),

[0554] [Chemical formula] The compound of formula (XV) is either of formula (XVa) or formula (XIVb),

[0555] [Chemical formula] The oligonucleoside comprises an RNA duplex including a first strand 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 strand and the second strand has 5' and 3' ends, and (i) in the case of formula (XVa), the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand, or (ii) in the case of formula (XVb), the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand. The method according to claim 69, dependent on claims 70 to 73.

[0556] 76. A compound of formula (XII),

[0557]

Chemical formula

[0558] 77. A compound of formula (XIIa).

[0559]

Chemical formula

[0560] 78. A compound of formula (XIIb).

[0561] [ka]

[0562] 79. A compound of formula (XIIc).

[0563] [ka]

[0564] 80. A compound of formula (XIId).

[0565] [ka]

[0566] 81. A compound of formula (XIII):

[0567] [ka] During the ceremony, R1, in each occurrence, is independently selected from the group consisting of hydrogen, methyl, and ethyl; m is an integer from 1 to 6; n is an integer from 1 to 10; compound.

[0568] 82. A compound of formula (XIIIa).

[0569] [ka]

[0570] 83. A compound of formula (XIIIb).

[0571] [ka]

[0572] 84. A compound of formula (XIV),

[0573] [Chemical formula] 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 each independently an integer from 0 to 4, provided that s, t, and v are not all 0 at the same time, a compound.

[0574] 85. A compound of formula (XIVa).

[0575] [Chemical formula]

[0576] 86. A compound of formula (XIVb).

[0577] [Chemical formula]

[0578] 87. A compound of formula (XV),

[0579] [Chemical formula] wherein, R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl at each occurrence, 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 are not both 0 at the same time, Z is an oligonucleoside moiety, Compound.

[0580] 88. A compound of formula (XVa).

[0581]

Chemical formula

[0582] 89. A compound of formula (XVb).

[0583]

Chemical formula

[0584] 90. Use of a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, for preparing a compound according to any one of propositions 76, 81 to 84, 87.

[0585] 91. Use of a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, wherein R2 = F, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, for preparing a compound according to proposition 85.

[0586] 92. Use of a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, wherein R2 = OH, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, for preparing a compound according to proposition 86.

[0587] 93. Use of the compound according to any one of Propositions 20, 25, 27, 29, 54, 56, and / or the composition according to any one of Propositions 30, 31, 57, 58 for preparing the compound according to Proposition 77.

[0588] 94. Use of the compound according to any one of Propositions 20, 25, 28, 29, 55, 56, and / or the composition according to any one of Propositions 30, 31, 57, 58 for preparing the compound according to Proposition 78.

[0589] 95. Use of the compound according to any one of Propositions 21, 26, 32, 34, 59, 61, and / or the composition according to any one of Propositions 35, 36, 62, 63 for preparing the compound according to Proposition 79.

[0590] 96. Use of the compound according to any one of Propositions 21, 26, 33, 34, 60, 61, and / or the composition according to any one of Propositions 35, 36, 62, 63 for preparing the compound according to Proposition 80.

[0591] 97. Use of the compound according to any one of Propositions 20, 25, 27 to 29, 54 to 56, and / or the composition according to any one of Propositions 30, 31, 57, 58 for preparing the compound according to Proposition 88.

[0592] 98. Use of the compound according to any one of Propositions 21, 26, 32 to 34, 59 to 61, and / or the composition according to any one of Propositions 35, 36, 62, 63 for preparing the compound according to Proposition 89.

[0593] 99. A compound or composition obtainable or capable of being obtained by the method according to any one of Propositions 68 to 75.

[0594] 100. A pharmaceutical composition comprising a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0595] 101. A compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63 for use in therapy.

[0596] In another aspect, the present invention can be applied to the compounds, methods, compositions, or uses of item numbers 1 to 56 below (references to any formula of the items refer only to the formulas defined within items 1 to 56. Such formulas are reproduced in Figure 7).

[0597] 1. The following structure:

[0598]

Chemical formula

[0599] 2. The compound according to item 1, wherein s is an integer selected from 4 to 12.

[0600] 3. The compound according to item 2, wherein s is 6.

[0601] 4. The compound according to any one of items 1 to 3, wherein r is an integer selected from 4 to 14.

[0602] 5. The compound according to item 4, wherein r is 6.

[0603] 6. The compound according to item 4, wherein r is 12.

[0604] 7. The compound according to item 5, which is dependent on item 3.

[0605] 8. The compound according to item 6, which is dependent on item 3.

[0606] 9. Z is

[0607]

Chemical formula

[0608] 10. The compound according to any one of items 1 to 9, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0609] 11. The RNA compound according to item 10, which comprises an RNA duplex containing a first strand 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, and each of the first strand and the second strand has 5' and 3' ends.

[0610] 12. The compound according to item 11, preferably also dependent on items 3 and 6, wherein the RNA compound is attached to an adjacent phosphate at the 5' end of its second strand.

[0611] 13. The RNA compound is the compound according to item 11, which is attached to the adjacent phosphate at the 3'-end of its second strand, preferably also dependent on items 3 and 5.

[0612] 14. A compound of formula (II * ), preferably dependent on item 12.

[0613]

Chemical formula

[0614] 15. A compound of formula (III * ), preferably dependent on item 13.

[0615]

Chemical formula

[0616] 16. The oligonucleoside is a compound defined in any of items 1 to 15, comprising an RNA duplex further comprising one or more riboses with a modified 2'-position, preferably a plurality of riboses with a modified 2'-position.

[0617] 17. The modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy, for the compound according to item 16.

[0618] 18. The oligonucleoside is a compound according to any of items 1 to 17, further comprising one or more degradation protection moieties at one or more termini.

[0619] 19. The one or more deprotection moieties are not present at the ends 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 in the distal strand of the same chain as the end carrying the linker / ligand moiety, the compound according to item 18.

[0620] 20. The ligand moiety illustrated in formula (I * ) of item 1 comprises one or more ligands, the compound according to any one of items 1 to 19.

[0621] 21. The ligand moiety illustrated in formula (I * ) of item 1 comprises one or more carbohydrate ligands, the compound according to item 20.

[0622] 22. The one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides, the compound according to item 21.

[0623] 23. 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, the compound according to item 22.

[0624] 24. The one or more carbohydrates comprise one or more N-acetyl-galactosamine moieties, the compound according to item 23.

[0625] 25. The compound according to item 24, comprising two or three N-acetylgalactosamine moieties.

[0626] 26. The one or more ligands are attached in a linear or branched configuration, the compound according to any of the preceding items.

[0627] 27. The compound according to item 26, wherein the one or more ligands are attached as a bifurcated or trifurcated branched chain structure.

[0628] 28. The moiety shown in the formula (I * ) of item 1:

[0629]

Chemical formula

[0630]

Chemical formula

[0631]

Chemical formula

[0632]

Chemical formula

[0633] 29. The formula (I in item 1 * ) of the said part shown in:

[0634]

Chemical formula

[0635]

Chemical formula

[0636] 30. The compound according to item 28 or 29, where a = 2.

[0637] 31. The compound according to item 28 or 29, where a = 3.

[0638] 32. The compound according to item 28, where b = 3.

[0639] 33. The compound of formula (VIII * ).

[0640]

Chemical formula

[0641] 34. The compound of formula (IX * ).

[0642]

Chemical formula

[0643] 35. The compound according to item 33 or 34, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0644] 36. The compound according to item 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0645] 37. The compound according to any one of items 33 to 36, wherein the oligonucleoside further comprises one or more deprotecting moieties at one or more termini.

[0646] 38. 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 end of the same strand as the terminus carrying the linker / ligand moiety. The compound according to item 37.

[0647] 39. The oligonucleoside comprises an RNA duplex comprising a first strand 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 strand and the second strand 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.

[0648] 40. The oligonucleoside comprises an RNA duplex comprising a first strand 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 strand and the second strand has 5' and 3' termini, and the RNA duplex is attached to an adjacent phosphate at the 3' terminus of its second strand. The compound according to item 34.

[0649] 41. A method for preparing a compound according to any one of items 1 to 40, the method comprising reacting a compound of formula (X * ) and (XI * ):

[0650]

Chemical formula

[0651] 42. The compound of formula (X * ) is of formula (Xa * ),

[0652]

Chemical formula

[0653]

Chemical formula

[0654] 43. The compound of formula (X * ) is of formula (Xb * ),

[0655]

Chemical formula

[0656]

Chemical formula

[0657] 44. The compound of formula (XIa * ) is of formula (XIb * ),

[0658]

Chemical formula

[0659] 45. Formula (X * ):

[0660]

Chemical formula

[0661] 46. A compound of formula (Xa * ).

[0662]

Chemical formula

[0663] 47. A compound of formula (Xb * ).

[0664]

Chemical formula

[0665] 48. A compound of formula (XI * ):

[0666]

Chemical formula

[0667] 49. A compound of formula (XIa * ).

[0668]

Chemical formula

[0669] 50. A compound of formula (XIb * ).

[0670] [Chemical]

[0671] 51. Use of a compound according to any one of items 45 and 48 to 50 for preparing a compound according to any one of items 1 to 40.

[0672] 52. Use of the compound according to item 46 for preparing a compound according to any one of items 6, 8 to 14, 16 to 33, and 35 to 40.

[0673] 53. Use of the compound according to item 47 for preparing a compound according to any one of items 5, 7, 9 to 13, 15 to 32, and 34 to 40.

[0674] 54. A compound or composition obtainable or obtained by a method according to any one of items 41 to 44.

[0675] 55. A pharmaceutical composition comprising a compound according to any one of items 1 to 40 together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0676] 56. A compound according to any one of items 1 to 40 for use in therapy. Examples

[0677] The present invention will be more fully understood by reference to the following examples. However, such examples 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 will be suggested to those skilled in the art based on them, and it is understood that they are included within the spirit and scope of the present application and within the scope of the appended claims. [Examples]

[0678] Target identification Background Genome-wide association (GWAS) studies aim to discover statistical associations between genetic variations (genotypes) in chromosomal DNA and an individual's physical or functional traits (phenotypes). There are several types of genetic variability, and the most commonly studied in GWAS among them are single nucleotide polymorphisms (SNPs), where individual nucleotides in the background DNA sequence of the genome can differ between individuals. Such SNPs can potentially lead to changes in gene or protein function, either directly (in the coding region of a gene) or indirectly (through effects on gene regulation). The mapping of SNPs to genes is not always 1:1; there can also be cases of many-to-one or one-to-many. GWAS studies are used in drug discovery to identify genes with (either positive or negative) mutations that correlate with the risk of developing certain diseases such as type 2 diabetes or atherosclerosis, or with certain outcomes that are consequences of such diseases, such as stroke or myocardial infarction. In some cases, by identifying such genes, clinically useful drug targets can be obtained, if most of the risk is concentrated in a single variant or if multiple variants map to a single gene. However, this is rather the exception than the rule.

[0679] In the case of complex (multifactorial) diseases such as diabetes, a more typical result of GWAS analysis is a long list of genes that are presumed to be correlated with the disease under investigation, but each gene in such a list has only a very small risk. The way to use such a long list of genes with weak associations to elucidate biological mechanisms and drive drug target discovery is a central problem at issue. In addition, typically, there is considerable uncertainty about mapping SNPs with weak underlying correlations to the genes they are associated with and about mapping the relationships between genes, so as a result, the underlying biological properties are opaque. In such a situation, the identification of clinically successful drug targets is very difficult and usually fails.

[0680] The inventors used network analysis techniques to analyze such "noisy" GWAS (and other omics analysis) gene lists (which may contain hundreds of weakly correlated genes and many mapping errors) to identify the underlying biological characteristics driving complex disease risks and to discover drug targets that could not otherwise be found by conventional methods, and developed a unique computational method for this purpose.

[0681] To achieve this, the inventors use a unique network analysis technique that assigns multiple genes to a smaller number of driver processes and enables the discovery of potent drug targets from such processes.

[0682] As described above, this technique utilizes information that is usually ignored in standard analyses, namely the known and predicted (using their own methods) interactions between genes (and proteins), as well as the prediction of other genes with which the GWAS (or other omics analysis) gene set, the so-called "hidden players", further interact.

[0683] Methods for identifying processes and targets The first step is to hypothesize that the dysfunctions correlated with "disease" should not be viewed at the level of individual genes. Rather, each gene belongs to a set whose members cooperate in a cooperative network module of interacting proteins. The network gives rise to biological processes, and it is the dysfunction at the level of this process or network module that should be considered the driver of risk.

[0684] Each protein within the network affected by SNPs contributes slightly to the overall dysregulation of the network module that controls the biological process. Also, network modules can interact to cause dysfunctions even at a more gross level of the tissue.

[0685] Therefore, after performing a detailed mapping, protein-coding genes were selected from the gene list. Using an internal curated database of all possible protein-protein interactions (derived from external experiments) and an internal "network construction" algorithm, a series of feasible networks were generated that included the maximum number of proteins in the list and the minimum number of complementary additional "hidden player" proteins. This algorithm uses pathways constrained by protein-protein interaction (PPI) data and attempts to find the optimal way to connect protein-coding genes by complementing missing proteins according to a "cost function".

[0686] This process captures the relationships between protein-coding genes within the GWAS list and adds other proteins ("hidden players") calculated to be involved in the same process.

[0687] In this way, multiple small effects are integrated across one or more networks to generate a larger effect.

[0688] The second step is to hypothesize that the connection patterns within such networks are important in determining the effects of gene dysfunction. This information is typically not readily available and is usually ignored in conventional analyses.

[0689] Using the networks obtained in the first step, a functional enrichment analysis was performed. The functional enrichment analysis incorporates information on the complementary hidden players and the connection patterns of the proteins in addition to the overlaps, thus differing from conventional approaches. That is, the relationships between the protein-coding genes within the GWAS list and the "hidden players" were used to identify the pathways important for the structure of the network.

[0690] Therefore, an internal curated pathway database that defines a set of proteins associated with a specific biological process was used. Such sets of proteins were then tested against the network by measuring the "structural impact" that the removal of common proteins would have on the network and assigning an "impact value" that depends on the specific wiring pattern of the network. Further statistical control was implemented to ensure that any bias in the statistical properties of the proteins within the GWAS set was properly controlled.

[0691] The third step is to hypothesize that the gene list associated with the dysregulated function is incomplete because of the compound errors and uncertainties outlined above, and in addition, mutations in some of the important proteins may not be tolerated because of their potential severity. Therefore, it is necessary to "complement" what is missing.

[0692] In the next step, the "network-enriched" pathways from the above analysis were plotted in two-dimensional space, where each pathway is represented by a point and the proximity of the points is a measure of the similarity of the pathways (see Figure 8). The pathway data was enhanced by using a search algorithm and the above PPI database and adding additional members that are "close in network space" according to another cost function. This made it possible to compare pathways that do not share many proteins but share "neighbors". Unsupervised machine learning techniques were used to cluster similar pathways. The biological functions of such clusters (processes associated with risk) were determined by interpretation by experts in pathway annotation and protein annotation.

[0693] In a further step, using an internal proprietary database of protein - protein relationships that includes the "directionality" of the interactions, a directed network model was reconstructed from selected clusters of pathway - protein sets that represent biological processes associated with disease risk. This direction information was derived from a series of public and internal databases complemented by directionality estimated from natural language processing of the text of scientific publications. A network construction algorithm using these information sources was used to construct a "directed" model of important biological processes.

[0694] Subsequently, proprietary analysis techniques were applied to the network model to identify pharmacologically promising targets within the network. Their knockdown would have a significant impact on the network and, by extension, on the modeled biological functions. Such algorithms extensively utilize direction information and hierarchical relationships to identify targets with a set of specific characteristics that would thereby make them good siRNA targets. The targets were then further filtered, if necessary, by protein class and hepatocyte specificity.

[0695] Identification of important processes and siRNA drug targets in type 2 diabetes The inventors used network biology techniques to create a network model of type 2 diabetes. The network model is designed to capture all the important proteins involved in the process, as well as their connectivity and, importantly, the direction of information flow between protein pairs.

[0696] The inventors analyzed such network models using a unique analysis method. In such a method, directional information is used to capture important "target" characteristics such as whether a protein is an information integration factor, whether it is an important pathway for information to other parts of the network, whether it is an influencing factor for important proteins, and the degree to which an influencing factor is affected or affects other proteins (based on the absolute and relative numbers and directions of inputs and outputs). Directional information also makes it possible to estimate the hierarchical relationship between proteins. Proteins that are higher in the hierarchy and have a particular characteristic may be more preferable than proteins that otherwise have similar characteristics. By comparing the relative specificity and magnitude of each characteristic with other characteristics, the inventors were able to score and rank proteins in terms of their target suitability.

[0697] The ability to characterize such target characteristics from the perspective of network relatedness makes it possible to judge the selectivity and magnitude of the effects in a selected situation, and thus the suitability of each for a given indication.

[0698] As a result, the inventors were able to identify targets that would lead to an improvement in the treatment of type 2 diabetes. The analysis used for this purpose was specially adjusted to find new and non-obvious targets for which knockdown by GalNAc-siRNA in hepatocytes would be beneficial for the treatment of diabetes.

[0699] For that purpose, the inventors utilized a large-scale GWAS meta-analysis of 898,930 individuals, 9% of whom were diabetic patients (Mahajan et al., Nature Genetics, 2018, Vol. 50, pp. 1505-1513).

[0700] From that GWAS meta-analysis, the inventors created a list of 257 genes derived from 403 different associated signals that have a weak correlation with the onset risk of type 2 diabetes. The 257 genes were subdivided into the categories of Figure 9.

[0701] Using the unique network analysis method described above, the present inventors were able to identify a specific biological process, "post-translational modification by glycosylation," that was significantly associated with type 2 diabetes risk in both normal and obese individuals. This process could not be identified by standard "functional enrichment" methods and was not identified by the authors of the meta-analysis (Mahajan et al., Nature Genetics, 2018, Vol. 50, pp. 1505-1513).

[0702] The present inventors were also able to demonstrate the recovery of known diabetes risk-related processes using network-aware techniques and an increase in the sensitivity of this method (see Figure 10).

[0703] Using a number of unique methods, the present inventors reconstructed the network model of this process and, using their analytical theory, ranked individual hepatocyte genes according to the predicted pharmacological effects and compatibility with GalNAc-mediated siRNA knockdown, and identified important target genes.

[0704] By this method, the present inventors were able to identify three hepatocyte-expressed genes encoding secretase products. Among them, B4GALT1 is the highest-ranked hepatocyte-expressed target in this analysis. A number of proteins were ranked in the upper quartile, but only three met the selection criteria of being hepatocyte-expressed, secreted, and an enzyme (Figure 11).

Example

[0705] Synthesis of Tether 1 Basic experimental conditions: Thin layer chromatography (TLC) was performed on silica-coated aluminum plates using Macherey-Nagel's 254 nm fluorescent indicator. The compounds were visualized under UV light (254 nm) or after spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to Stahl (from Sigma-Aldrich) and subsequent heating. Flash chromatography was carried out on a Biotage Isolera One flash chromatography instrument equipped with a dual variable UV wavelength detector (200 - 400 nm) using Biotage Sfar silica 10, 25, 50, or 100 g columns (Uppsala, Sweden).

[0706] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich and the solvents were purchased from Carl Roth GmbH+Co.KG. D-Galactosamine pentaacetate was purchased from AK scientific.

[0707] HPLC / ESI-MS was performed using a Dionex UltiMate 3000RS UHPLC device and a Thermo Scientific MSQ Plus mass spectrometer with a Waters Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1×100 mm) at 60 °C. 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% to 100% B over 15 minutes was used at a flow rate of 0.4 mL / min. Detector and conditions: Corona ultra charged aerosol detection (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0708] 1 H and 13 C NMR spectra were recorded at room temperature on a 500 MHz ( 1 H NMR) and 125 MHz ( 13Recorded on a Varian spectrometer (for 13C NMR). Chemical shifts are indicated in ppm relative to the solvent residual peak (CDCl3 - 1 1H NMR: δ 7.26 ppm and 13 13C NMR δ 77.2 ppm; DMSO-d6 - 1 1H NMR: δ 2.50 ppm and 13 13C NMR δ 39.5 ppm). Coupling constants are indicated in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[0709] Synthetic route of the conjugate building block TriGalNAc_tether1:

[0710]

Chem.

[0711] Preparation of Compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 equiv) was added. The reaction 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. It 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)).

[0712]

Chem.

[0713] Preparation of Compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 equiv) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 equiv) were dissolved in anhydrous DCM (40 mL) under argon, and molecular sieves 3 Å (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. 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, 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).

[0714] [Chemistry]

[0715] 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 volume / volume), and Pd / C (100 mg) was added. The reaction mixture was degassed by a vacuum / argon cycle (3 times) 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)). This compound was used without further purification. MS: C 20 H 34 N2O 11 Calculated value for, 478.2. Found 479.4.

[0716] [Chemistry]

[0717] 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 volume / volume), 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 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% ethyl acetate in cyclohexane, 12 CV) to give the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: 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).

[0718]

Chem.

[0719] 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 the TFA salt (0.183 g, 98%). This compound was used without further purification. MS: C 21 H 29 N 11 Calculated value, 471.6. Measured value 472.4.

[0720]

Chem.

[0721] 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 this solution, and the reaction mixture 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 product was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 14 CV). The product was obtained as a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: 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).

[0722]

Chem.

[0723] 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 by vacuum / argon cycling (3 times) 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 resulting residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellow oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 calculated value of, 1718.8. Measured value 1719.3.

[0724]

Chem.

[0725] 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 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 16 CV). The product was obtained as a white solid (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM)). MS: C 15 H 23 Calculated for C

[0726]

Chemical Structure

[0727] 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 (gradient elution: 0 - 10% MeOH in DCM, 20 CV) to give the title compound as a white, fluffy solid (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)). MS: C 84 H 137 N 11 O 41 Calculated for, 1957.1. Found 1959.6.

[0728] Conjugation of the tether 1 with the siRNA strand: Monofluoro cyclooctyne (MFCO) conjugation at the 5'- or 3'-end 5'-end MFCO conjugation

[0729]

Chem.

[0730]

Chem.

[0731] Basic 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 (volume / volume). To this solution, 1 molar equivalent of a DMF solution of 35 mM MFCO-C6-NHS ester (Berry & Associates, catalog number LK4300) was added. The reaction was carried out at room temperature, and after 1 hour, another 1 molar equivalent of the MFCO solution was added. The reaction was allowed to proceed for a further 1 hour and monitored by LC / MS. To achieve quantitative consumption of the starting material, at least 2 molar equivalents in excess of the MFCO NHS ester reagent were required relative to the amino-modified oligonucleotide. 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) using an Akta Pure instrument (GE Healthcare).

[0732] 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.

[0733] Fractions containing full-length conjugate oligonucleotides were pooled and precipitated with 3M NaOAc, pH 5.2 and 85% ethanol in a freezer, and the collected pellet was dissolved in water. The sample was desalted by size-exclusion chromatography, concentrated using a speed-vac concentrator, and the conjugate oligonucleotide was obtained with an isolated yield of 40 - 80%. 5’-GalNAc-T1 conjugate

[0734]

Chemical formula

[0735]

Chemical formula

[0736] Basic procedure for TriGalNAc conjugation: The MFCO-modified single-strand was dissolved in water at 2000 OD / mL, and 1 equivalent of a DMF solution of compound 12 (10 mM) was added to this solution. The reaction was carried out at room temperature, and after 3 hours, 0.7 molar equivalent of the compound 12 solution was added. The reaction was allowed to proceed overnight and monitored for completion by LCMS. The conjugate was diluted 15-fold with water, filtered through a Sartorius 1.2 μm filter, and then purified by RP HPLC on an Akta Pure instrument (GE Healthcare).

[0737] RP HPLC purification was carried out using a Waters XBridge C18 Prep 19×50 mm column. 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.

[0738] Fractions containing full-length conjugate oligonucleotides were pooled and precipitated with 3M NaOAc, pH 5.2 and 85% ethanol in a freezer. 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 such protecting groups was verified by LC-MS.

[0739] The conjugate was desalted by size-exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to obtain conjugate oligonucleotides in an isolated yield of 50 - 70%.

[0740] The following scheme further shows the synthetic route.

[0741]

Chemical formula

[0742]

Chemical formula

[0743]

Chemical formula

[0744]

Chemical formula

[0745]

Chemical formula

Example

[0746] Double-stranded annealing To generate the desired siRNA duplex, the two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixture was placed in a water bath at 70 °C for 5 minutes, followed by allowing cooling to ambient temperature within 2 hours. The duplex was lyophilized for 2 days and stored at -20 °C.

[0747] The duplex was analyzed by analytical SEC HPLC on a Superdex™ 75 Increase 5 / 150 GL column 5 × 153 - 158 mm (Cytiva) using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC instrument. The mobile phase consisted of 1× PBS containing 10% acetonitrile. An isocratic gradient was run at room temperature for 10 minutes at a flow rate of 1.5 mL / min. 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).

Example

[0748] Synthesis of Tether 2 Basic experimental conditions: Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using Macherey-Nagel's 254 nm fluorescent indicator. Compounds were visualized under UV light (254 nm) or by spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent according to Stahl (from Sigma-Aldrich), followed by heating. Flash chromatography was performed on a Biotage Isolera One flash chromatography instrument equipped with a dual variable UV wavelength detector (200 - 400 nm) using Biotage Sfar silica 10, 25, 50, or 100 g columns (Uppsala, Sweden).

[0749] All humidity-responsive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and the solvents were purchased from Carl Roth GmbH+Co.KG. D-Galactosamine pentaacetate was purchased from AK scientific.

[0750] HPLC / ESI-MS was performed using a Dionex UltiMate 3000RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer with a Waters Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1×100 mm) at 60 °C. The solvent system consisted of solvent A, which was H2O containing 0.1% formic acid, and solvent B, which was acetonitrile (ACN) containing 0.1% formic acid. A gradient of 5% to 100% B over 15 minutes was used at a flow rate of 0.4 mL / min. Detector and conditions: Corona ultra charged aerosol detection (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0751] 1 H and 13 C NMR spectra were recorded at room temperature on a Varian spectrometer operating at 500 MHz ( 1 H NMR) and 125 MHz ( 13 C NMR). Chemical shifts are reported in ppm relative to the solvent residual peak (CDCl3 - 1 H NMR: δ 7.26 ppm and 13 C NMR δ 77.2 ppm; DMSO-d6 - 1 H NMR: δ 2.50 ppm and 13 C NMR δ 39.5 ppm). Coupling constants are reported in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[0752] Synthetic route of the conjugate building block TriGalNAc_tether2:

[0753]

Chem.

[0754] Preparation of Compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 equiv) was added. The reaction mixture was stirred at room temperature for 3 hours. 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. It 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)).

[0755]

Chem.

[0756] 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, 10 CV) to give the title product as a light yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: C 20 H32 N4O 11 Calculated value, 504.21. Measured value 505.4. 1 H NMR (500 MHz, CDCl3) δ 6.21 - 6.14 (m, 1H), 5.30 (dd, J = 3.4, 1.1 Hz, 1H), 5.04 (dd, J = 11.2, 3.4 Hz, 1H), 4.76 (d, J = 8.6 Hz, 1H), 4.23 - 4.08 (m, 3H), 3.91 - 3.80 (m, 3H), 3.74 - 3.59 (m, 9H), 3.49 - 3.41 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.97 (d, J = 4.2 Hz, 6H). 13 C NMR (125 MHz, CDCl3) δ 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).

[0757]

Chem.

[0758] 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 volume / volume), and Pd / C (100 mg) was added. The reaction mixture was degassed by vacuum / argon cycle (3 times) 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 obtain the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). This compound was used without further purification. MS: C 20 H 34 N2O 11Calculated value: 478.2, measured value: 479.4.

[0759] [Chem.]

[0760] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 equivalent) was dissolved in a mixture of DCM / water (40 mL, 1:1 volume / volume), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 equivalent) was added with vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 equivalents) was added dropwise to the mixture, and the reaction mixture was stirred at room temperature for 24 hours. 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% ethyl acetate in cyclohexane, 12 CV) to obtain the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: C 33 H 53 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).

[0761] [Chemistry]

[0762] 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 the TFA salt (0.183 g, 98%). This compound was used without further purification. MS: C 21 H 29 NO 11 Calculated value for, 471.6. Found 472.4.

[0763] [Chemistry]

[0764] 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 this solution, and the reaction mixture 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 product was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 14 CV). The product was obtained as a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: C81 H 125 N7O 41 Calculated value, 1852.9. Measured value 1854.7. 1 H NMR (500 MHz, DMSO-d6) δ 7.90 - 7.80 (m, 10H), 7.65 - 7.62 (m, 4H), 7.47 - 7.43 (m, 3H), 7.38 - 7.32 (m, 8H), 5.24 - 5.22 (m, 3H), 5.02 - 4.97 (m, 4H), 4.60 - 4.57 (m, 3H), 4.07 - 3.90 (m 10H), 3.67 - 3.36 (m, 70H), 3.23 - 3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80 - 1.78 (m, 17H). 13 C 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).

[0765]

Chem.

[0766] 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 by vacuum / argon cycle (3 times) 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 yellow oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 Calculated for, 1718.8. Found 1719.3.

[0767]

Chem.

[0768] 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, 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.

[0769]

Chem.

[0770] 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 vacuum / argon cycle (3 times) 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 obtained residue was dried under high vacuum overnight. The residue was used for conjugation with oligonucleosides without further purification (0.28 g, quantitative yield). MS: C 81 H 131 N7O 42 Calculated value of, 1874.9. Measured value 1875.3.

[0771] Conjugation of Tether 2 with siRNA strand: TriGalNAc Tether 2 (GalNAc-T2) conjugation at the 5'-end or 3'-end 5'-GalNAc-T2 conjugate

[0772]

Chemical Structure

[0773]

Chemical Structure

[0774] Preparation of TriGalNAc Tether 2 NHS ester: To a solution of carboxylic acid Tether 2 (Compound 15, 227 mg, 121 μmol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol) were added. The solution was stirred at room temperature for 18 hours and used for the subsequent conjugation reaction without purification.

[0775] Basic 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 (volume / volume), and to this solution was added a 1 molar equivalent solution of tether 2 NHS ester (57 mM) in DMF. The reaction was carried out at room temperature and after 1 hour another 1 molar equivalent of NHS ester solution was added. The reaction was allowed to proceed for a further 1 hour and the progress of the reaction was monitored by LCMS. To achieve quantitative consumption of the starting material, at least a 2 molar equivalent excess of NHS ester reagent was required relative to the amino-modified oligonucleotide. The reaction mixture was diluted 15-fold with water, filtered through a Sartorius 1.2 μm filter, and then purified by reverse phase (RP HPLC) on an Akta Pure (GE Healthcare) instrument.

[0776] Purification was carried out using a Waters XBridge C18 Prep 19×50 mm column. Buffer A was 100 mM TEAA pH 7 and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60 °C were used. A UV trace at 280 nm was recorded. A gradient of 0 - 100% B was used within 60 column volumes.

[0777] Fractions containing the full-length conjugate oligonucleotide were pooled and precipitated with 3M NaOAc, pH 5.2 and 85% ethanol in a freezer and then dissolved in water at 1000 OD / mL. O-Acetate was removed with 20% aqueous ammonium hydroxide until completion (monitored by LC-MS).

[0778] The conjugate was desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to obtain the conjugate oligonucleotide in 60 - 80% isolated yield.

[0779] The conjugate was characterized by HPLC-MS analysis using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics) and a 2.1×50 mm XBridge C18 column (Waters). Buffer A was 16.3 mM triethylamine, 100 mM HFIP in 1% aqueous MeOH, 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.

[0780] The following scheme further shows the synthetic route.

[0781] [Chemical formula]

[0782] [Chemical formula]

[0783] [Chemical formula]

[0784] [Chemical formula] [Examples]

[0785] Double-strand annealing To generate the desired siRNA double strand, the 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, followed by allowing cooling to ambient temperature within 2 hours. The double strand was lyophilized for 2 days and stored at -20 °C.

[0786] The double-strand was analyzed by analytical SEC HPLC using a Superdex™ 75 Increase 5 / 150 GL column 5 × 153 - 158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC instrument. The mobile phase consisted of 1×PBS containing 10% acetonitrile. An isocratic gradient was run at a flow rate of 1.5 mL / min for 10 minutes 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).

Example

[0787] Alternative synthetic route for conjugate building block TriGalNAc-tether 2:

[0788]

Chem.

[0789]

Chem.

[0790] Conjugation of tether 2 with siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at the 5'-end or 3'-end Conjugation conditions

[0791]

Chem.

[0792]

Chemical formula

[0793]

Chemical formula

Example

[0794] Solid-phase synthesis method: Scale ≤ 1 μMOL The synthesis of the siRNA sense and antisense strands was carried out on a commercially available solid support made of porous controlled glass with a universal linker (universal CPG, loading 40 μmol / g; LGC Biosearch or Glen Research) using a MerMade192X synthesizer.

[0795] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[0796] 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.

[0797] 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-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0798] All phosphoramidites, except for 2'-O-methyl-uridine phosphoramidite dissolved in DMF / MeCN (1:4, volume / volume), 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 of phosphorothioate linkages was carried out using 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 volume / volume. 5-Ethylthiotetrazole (ETT) at 0.25 M mM in acetonitrile was used as the activator solution.

[0799] Inverted deoxy base phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[0800] In each cycle, DMT was removed with the deblocking solution, 3% TCA in DCM (DNAchem).

[0801] The coupling time was 180 seconds. The oxidant contact time was set to 80 seconds, and the thiolation time was 2 * 100 seconds.

[0802] At the end of the synthesis, the oligonucleotide was cleaved from the solid support using an NH4OH:EtOH solution 4:1 (volume / volume) (TCI) at 45 °C for 20 hours. The solid support was then filtered, the filter was washed well with H2O, and the volume of the mixed solution was reduced by evaporation under reduced pressure.

[0803] The oligonucleotide was processed 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.

[0804] The identity of a single-strand was evaluated by MS ESI, and then annealed in water to form the final double-stranded siRNA. The double-strand purity was evaluated by size exclusion chromatography.

Example

[0805] Solid-phase synthesis method: scale ≥ 5 μMOL The synthesis of the siRNA sense strand and the antisense strand was carried out on a commercially available solid support made of porous controlled glass with a universal linker (universal CPG, loading 40 μmol / g; LGC Biosearch or Glen Research) at a scale of 5 μmol using a MerMade12 synthesizer. The sense strand for the purpose of 3'-conjugation was synthesized at 12 μmol on a 3'-PT-amino modified substance C6 CPG 500 Å solid support (LGC) with a loading of 86 μmol / g.

[0806] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[0807] 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.

[0808] 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.

[0809] 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).

[0810] All phosphoramidites except 2'-O-methyl-uridine phosphoramidite dissolved in DMF / MeCN (1:4, volume / volume) 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 of phosphorothioate linkages was carried out using 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 volume / volume. 5-Ethylthiotetrazole (ETT) at 0.25 M mM in acetonitrile was used as the activator solution.

[0811] In each cycle, the DMT was removed with the deblock solution, 3% TCA (DNAchem) in DCM.

[0812] For the case of the chain synthesized with universal CPG, the 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.

[0813] For the case of the chain synthesized with 3’-PT-amino-modified substance C6 CPG, the coupling was carried out for * 150 seconds using 8 equivalents of amidite. The oxidation time was 47 seconds and the thiolation time was 250 seconds.

[0814] At the end of the synthesis, the oligonucleotide was cleaved from the solid support using a 4:1 (volume / volume) NH4OH:EtOH solution (TCI) at 45 °C for 20 hours. The solid support was then filtered, the filter was washed well with H2O, and the volume of the mixed solution was reduced by evaporation under reduced pressure.

[0815] The oligonucleotide was treated by ethanol precipitation from 1M sodium acetate to form the sodium salt.

[0816] The single-stranded oligonucleotide was purified by IP-RP HPLC using an Xbridge BEH C18 5μm, 130Å, 19×150mm (Waters) column with a 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.

[0817] The single-stranded purity and identity were evaluated by UPLC / MS ESI- using an Xbridge BEH C18 2.5μm, 3×50mm (Waters) column with a gradient of B in A. Mobile phase A: 100 mM HFIP, 5 mM TEA in water; Mobile phase B: 20% of mobile phase A: 80% acetonitrile (volume / volume).

[0818] The sense strand was conjugated according to the protocol provided in any of Examples 2, 4, 6.

[0819] Next, the sense strand and the antisense strand were annealed in water to form the final double-stranded siRNA, and the double-strand purity was evaluated by size exclusion chromatography.

[0820] 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 become apparent from the description and teachings herein. Such variations can be practiced without departing from the true scope and spirit of the present disclosure and are intended to fall within the scope of the present disclosure.

Example

[0821] B4GALT1 Pharmacological Research Through in silico computational biology analysis of ETX tissues, it was identified that B4GALT1, which encodes beta-1,4-galactosyltransferase 1, is a gene associated with type 2 diabetes (T2D) (see Example 1). The inventors here establish that B4GALT1 is a potential therapeutic target for T2D. To evaluate the validity of the hypothesis that significant knockdown of hepatic B4GALT1 mRNA reduces the plasma levels of LDL-c, fibrinogen, and fasting blood glucose, in silico-designed GalNAc-siRNAs targeting mouse hepatic B4GALT1 were synthesized and tested.

[0822] In Vitro Dose-Response Assay for Selecting Potent Molecules An in vitro dose-response assay was performed to measure gene knockdown in primary mouse hepatocytes (PMH), and 20 types of GalNAc-siRNAs targeting liver B4GALT1 were tested. Primary C57BL / 6 mouse hepatocytes (PMH) were freshly isolated by two-step collagenase liver perfusion. The cells were maintained in DMEM (Gibco-11995-092) supplemented with FBS, penicillin / streptomycin, HEPES, and L-glutamine. The cells were cultured at 37 °C in a 5% CO2 atmosphere in a humidified incubator. Within 2 hours after isolation, PMH were seeded at a density of 36,000 cells / well in a normal 96-well tissue culture plate. The dose-response analysis in PMH was performed by directly incubating the cells in a setting of free uptake of gymnosis at final GalNAc-siRNA concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM. In the control wells, the cells were incubated without GalNAc-siRNA. After culturing for 48 hours, the cells were harvested for RNA extraction. Total RNA was extracted using the RNeasy kit according to the manufacturer's instructions (Qiagen, Shanghai, China). After reverse transcription, real-time quantitative PCR was performed using ABI Prism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH. The expression of the target gene in each test sample was determined by relative quantification using the comparative Ct (ΔΔCt) method. In this method, the Ct difference (ΔCt) between the target gene and the housekeeping gene is measured. The formula is as follows: ΔCt = average Ct of B4GALT1 - average Ct of GAPDH, ΔΔCt = ΔCt (sample) - average ΔCt (untreated control), relative expression of target gene mRNA = 2 -ΔΔCt . Based on the results of the in vitro free uptake experiment, GalNAc-siRNAs showing good activity were selected, and an EC 50 was determined using a 10-point concentration curve (Figure 12).

[0823] In vivo pharmacology with four selected GalNAc-siRNAs The pharmacodynamic activities of four selected B4GALT1 GalNAc-siRNAs were measured in vivo. Twelve male C57BL / 6 mice were assigned to each of the GalNAc-siRNAs, ETXM619, ETXM624, ETXM628, and ETXM633. Five mice were assigned to the untreated control group. The mice were dosed subcutaneously with ETXM (10 mg / kg) on days 0, 3, and 7, defined as the day when the mice were first dosed. Three mice from each treatment group were sacrificed on days 3, 7, 10, and 14. After the dosing was completed, liver tissue and plasma samples were collected and further analyzed. Samples on day 3 were used to evaluate the single-dose effect of ETXM given on day 0. Samples on day 7 represent the repeated-dose effect of ETXM given on days 0 and 3. Similarly, samples on days 10 and 14 represent the repeated-dose effect of ETXM given on days 0, 3, and 7. Five mice assigned to the control group were sacrificed on day 14.

[0824] B4GALT1 gene knockdown in mouse liver The collected liver samples were used to measure the B4GALT1 mRNA knockdown level by RT-qPCR. After collection, each tissue was treated with RNAlater, stored at 4°C overnight, and then stored at -80°C until further analysis. To extract RNA, liver tissue was homogenized with TRIZOL. RNA samples adjusted to 400 ng / μL were reverse-transcribed into cDNA using the FastKing RT kit by TIANGEN. After the gDNA removal procedure, the purified cDNA samples were used for RT-qPCR. The RT-qPCR method and relative mRNA expression calculation were as described above. Figure 13 shows that all test substances exhibited a gene knockdown efficiency of >50% on days 3, 7, 10, and 14.

[0825] Final plasma collection and measurement of plasma biomarkers using a biochemical analyzer The final plasma samples were collected from the submandibular vein after a 4- to 5-hour fast. Blood samples were collected into heparin sodium-coated tubes and centrifuged at 7,000 g for 10 minutes at 4°C to obtain plasma samples. The plasma samples were used for the measurement of AST, ALT, albumin, ALP, BUN, CREA, TBIL, glucose, total cholesterol, LDL-c, HDL-c, triglyceride, and NEFA (free fatty acid) by a biochemical analyzer.

[0826] Measurement of plasma insulin and fibrinogen levels using an ELISA kit Blood samples were collected into K2EDTA-coated tubes and then centrifuged at 7,000 g for 10 minutes at 4°C to obtain plasma samples. Plasma insulin concentration was measured using a mouse insulin ELISA kit (Mercodia, 10-1247-01) according to the manufacturer's protocol. Fibrinogen plasma level was measured using a mouse fibrinogen antigen assay kit (Innovative Research, IMSFBGKTT).

[0827] B4GALT1 gene silencing effect in biomarker modulation The means of the untreated control group (n = 5) and the 14-day treatment group including the groups (n = 3 per group, total n = 12) administered subcutaneously with ETXM619, ETXM624, ETXM628, or ETXM633 on day 0, day 3, and day 7 were tested for equivalence under the null hypothesis by a two-sided t-test. Statistically significant differences were detected in the means of the efficacy biomarker readings, with a 18.8% decrease in LDL-C (p < 0.05), a 21.0% decrease in fasting blood glucose (p < 0.05), and a 29.6% decrease in fibrinogen (p < 0.01) (Figure 14).

Claims

1. A pharmaceutical composition comprising an inhibitor of B4GALT1 expression and / or function for use in the treatment of diabetes.

2. A pharmaceutical composition comprising a post-translational glycosylation inhibitor for use in the treatment of diabetes.

3. The pharmaceutical composition according to claim 1 or 2, wherein the inhibitor is an siRNA oligomer conjugated to one or more ligand portions.

4. The pharmaceutical composition according to claim 3, wherein the one or more ligand portions comprise one or more GalNAc ligands and / or one or more GalNAc ligand derivatives.

5. A post-translational glycosylation inhibitor, which is conjugated to one or more ligand moieties.

6. The inhibitor according to claim 5, wherein the inhibitor comprises one or more ligand-conjugated siRNA oligomers.

7. The inhibitor according to claim 5 or 6, wherein the one or more ligand portions comprise one or more GalNAc ligands and / or one or more GalNAc ligand derivatives.

8. The inhibitor or pharmaceutical composition according to claim 1, 2, or 5, wherein the target of the inhibitor is B4GALT1.

9. The inhibitor is an siRNA oligomer having a first chain and a second chain, i) The first strand of the siRNA has a length in the range of 15 to 30 nucleosides, and / or ii) The second strand of the siRNA has a length in the range of 15 to 30 nucleosides. The inhibitor or pharmaceutical composition according to claim 1, 2, or 5.

10. The inhibitor or pharmaceutical composition according to claim 9, wherein the second sense chain further comprises one or more debased nucleosides in the terminal region of the second chain, the debased nucleosides being connected to adjacent nucleosides via reverse internucleoside linkages.

11. The inhibitor or pharmaceutical composition according to claim 9, wherein one or more nucleosides of the first chain and / or the second chain are modified to form a modified nucleoside.

12. The inhibitor according to claim 5, formulated as a pharmaceutical composition having an excipient and / or a carrier.

13. A pharmaceutical composition comprising the inhibitor according to claim 5, in combination with a pharmaceutically acceptable excipient or carrier.

14. A pharmaceutical composition comprising the inhibitor according to claim 5, in combination with a pharmaceutically acceptable excipient or carrier, for use in the treatment of diabetes.

15. Use of B4GALT1 as a target to identify one or more therapeutic agents for the treatment of diabetes.

16. B4GALT1 for use as a diabetes biomarker.

17. Typically, B4GALT1 is used in vivo to predict susceptibility to diabetes by monitoring the sequence and / or expression and / or function levels of B4GALT1 in patient samples.

18. A method for predicting a patient's susceptibility to diabetes, (a) To detect the sequence and / or expression and / or function of B4GALT1 in the sample obtained from the patient, (b) Predict susceptibility to diabetes based on the sequence and / or expression and / or function of B4GALT1 in the sample obtained from the patient, A method that includes this.

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