Expression and / or function inhibitors

Abstract

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

Description

Technical Field

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

[0003] Inhibitors such as oligonucleotide / oligonucleoside 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 cause specific diseases. Gene silencing prevents the formation of proteins by inhibiting translation. Importantly, gene silencing agents are promising alternatives to traditional small molecule organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleotide / oligonucleoside compounds that prevent protein formation through gene silencing.

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

[0005] The present invention relates to inhibitors, such as oligomers such as nucleic acids (such as oligonucleotide / oligonucleoside compounds), and their use in the treatment and / or prevention of diseases.

[0006] In particular, there is still a need for suitable inhibitors to help prevent and / or treat diseases such as type 2 diabetes.

[0007] A mutation in the B4GALT1 gene that results in a 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 WO2018226560, and Montasser et al., Science 374, 1221–1227 (2021) December 3, 2021). It has been proposed to use siRNA that hybridizes to a sequence within the endogenous B4GALT1 gene and reduces the expression of B4GALT1 polypeptide in the cells of a subject as a means of treating subjects suffering from or prone to developing cardiovascular disease. Summary of the Invention

[0008] The present invention is defined in the claims and in particular relates to:

[0009] In one aspect, the present invention relates to inhibitors of post-translational glycosylation, such as inhibitors of B4GALT1 expression and / or function, wherein the inhibitor is conjugated to one or more ligand moieties.

[0010] In another aspect, the present invention relates to an inhibitor according to the present invention, wherein the inhibitor comprises an siRNA oligomer conjugated to one or more ligand moieties.

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

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

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

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

[0015] In another aspect, the present invention relates to an inhibitor used according to the present invention, which is an siRNA oligomer, typically conjugated to one or more ligand moieties.

[0016] In another aspect, the present invention relates to an inhibitor used according to the present invention, wherein the one or more ligand moieties comprise one or more GalNAc ligands, and / or one or more GalNAc ligand derivatives.

[0017] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used according to the present invention, wherein the target of the inhibitor is selected from B4GALT1.

[0018] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used according to the present invention, which is an siRNA oligomer having a first strand and a second strand, wherein:

[0019] i) The length of the first strand of the siRNA ranges from 15 to 30 nucleotides, preferably from 19 to 25 nucleotides, more preferably from 23 or 25 nucleotides; even more preferably from 23 nucleotides; and / or

[0020] ii) The length of the second strand of the siRNA ranges from 15 to 30 nucleotides, preferably from 19 to 25 nucleotides, more preferably from 21 nucleotides.

[0021] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the second sense strand further comprises one or more abasic nucleosides in the terminal region of the second strand, and wherein said one or more abasic nucleosides are linked to adjacent nucleosides by an inverse internucleoside linkage.

[0022] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the second strand comprises:

[0023] i two or more abasic nucleosides in the terminal region of the second strand; and / or

[0024] ii two or more abasic nucleosides in the 5' or 3' terminal region of the second strand; and / or

[0025] iii two or more abasic nucleosides in the 5' or 3' terminal region of the second strand, wherein said abasic nucleosides are present in an overhang as described herein; and / or

[0026] iv two or more consecutive abasic nucleosides in the terminal region of the second strand, wherein preferably, one such abasic nucleoside is the terminal nucleoside; and / or

[0027] v two or more consecutive abasic nucleosides in the 5' or 3' terminal region of the second strand, wherein preferably, one such abasic nucleoside is the terminal nucleoside in the 5' or 3' terminal region of the second strand; and / or

[0028] vi an inverse internucleoside linkage links at least one abasic nucleoside to an adjacent base nucleoside in the terminal region of the second strand; and / or

[0029] vii an inverse internucleoside linkage links at least one abasic nucleoside to an adjacent base nucleoside in the 5' or 3' terminal region of the second strand; and / or

[0030] viii an abasic nucleoside as the penultimate nucleoside, which is linked by an inverse bond to a nucleoside that is not the terminal nucleoside (referred to herein as the antepenultimate nucleoside); and / or

[0031] ix when reading the strand in the direction towards the terminal, the abasic nucleoside is two terminal nucleosides linked by a 5'-3' bond;

[0032] x when reading the strand in the direction towards the end containing the terminal nucleoside, the abasic nucleoside is two terminal nucleosides linked by a 3'-5' bond;

[0033] The xiiibases-free nucleosides serve as the terminal two positions, wherein the penultimate nucleoside is linked to the antepenultimate nucleoside via a reverse bond, and wherein the reverse bond is a 5-5’ reverse bond or a 3’-3’ reverse bond;

[0034] xiiibases-free nucleosides serve as the terminal two positions, wherein the penultimate nucleoside is linked to the antepenultimate nucleoside via a reverse bond, and wherein

[0035] (1) the reverse bond is a 5-5’ reverse bond, and when reading towards the end containing the terminal bases-free nucleoside and the penultimate bases-free nucleoside, the bond between the terminal bases-free nucleoside and the penultimate bases-free nucleoside is 3’5’; or

[0036] (2) the reverse bond is a 3-3’ reverse bond, and when reading towards the end containing the terminal bases-free nucleoside and the penultimate bases-free nucleoside, the bond between the terminal bases-free nucleoside and the penultimate bases-free nucleoside is 5’3’.

[0037] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the reverse internucleoside bond is located in the terminal region distal to the 5’-terminal region of the second strand, or in the terminal region distal to the 3’-terminal region of the second strand.

[0038] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the reverse internucleoside bond is a 3’3 reverse bond.

[0039] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the reverse internucleoside bond is a 5’5 reverse bond.

[0040] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein one or more nucleosides on the first strand and / or the second strand are modified to form modified nucleosides.

[0041] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the modification is a modification at the 2’-OH group of ribose, optionally selected from 2’-Me or 2’-F modifications.

[0042] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein counting from position 1 of the first strand, the first strand contains 2’-F at any position among position 14, position 2, position 6, or any combination thereof.

[0043] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein counting from position 1 of the second strand, the second strand contains 2’-F modification at position 7 and / or 9, and / or 11 and / or 13.

[0044] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein each of the first strand and the second strand comprises 2'-Me and 2'-F modifications.

[0045] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which is an siRNA, wherein the siRNA comprises at least one heat-destabilizing modification, which is suitably located at one or more positions from position 1 to position 9 of the first strand counted from position 1 of the first strand, and / or at one or more positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid.

[0046] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein counted from position 1 of the first strand, the siRNA comprises at least one heat-destabilizing modification at position 7 of the first strand.

[0047] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which is an siRNA, wherein counted from position 1 of the second strand, the siRNA comprises 3 or more 2'-F modifications at positions 7 to 13 of the second strand, for example, 4, 5, 6 or 7 2'-F modifications at positions 7 to 13 of the second strand.

[0048] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which is an siRNA, wherein counted from position 1 of the second strand, the second strand comprises at least 3, for example 4, 5 or 6 2'-Me modifications at positions 1 to 6 of the second strand.

[0049] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which is an siRNA, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3'-terminal region, preferably comprises the terminal nucleoside of the 3'-terminal region, or at least within 1 or 2 nucleosides of the terminal nucleoside of the 3'-terminal region.

[0050] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which is an siRNA, wherein the first strand comprises 7 consecutive 2'-Me modifications in the 3'-terminal region, preferably comprises the terminal nucleoside in the 3'-terminal region.

[0051] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the siRNA oligomer further comprises one or more phosphorothioate internucleoside bonds.

[0052] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein one or more phosphorothioate internucleoside linkages are respectively located between at least three consecutive positions in the 5' or 3' proximal end region of the second strand, wherein the proximal end region preferably adjoins the terminal region, and wherein the positions of the one or more abasic nucleosides of the second strand are as defined herein.

[0053] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein one or more phosphorothioate internucleoside linkages are respectively located between at least three consecutive positions in the 5' and / or 3' terminal region of the first strand, and wherein preferably the terminal positions of the 5' and / or 3' terminal region of the first strand are linked to their adjacent positions by phosphorothioate internucleoside linkages.

[0054] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the oligomer is siRNA, and the second strand of the siRNA is directly or indirectly conjugated to one or more ligand moieties, and wherein the ligand moieties are generally present in the terminal region of the second strand, preferably in its 3' terminal region.

[0055] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the ligand moiety comprises:

[0056] i) one or more GalNAc ligands; and / or

[0057] ii) one or more derivatives of GalNAc ligands; and / or

[0058] iii) one or more GalNAc ligands and / or GalNAc ligand derivatives conjugated to the siRNA via a linker.

[0059] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly conjugated to the 5' or 3' terminal region of the second strand of the siRNA oligomer, preferably in its 3' terminal region.

[0060] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, wherein the ligand moiety comprises:

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[0062] On the other hand, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which has the following structure:

[0063]

[0064] Wherein:

[0065] Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl and ethyl;

[0066] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro;

[0067] Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen and sulfur;

[0068] m is an integer from 1 to 6;

[0069] n is an integer from 1 to 10;

[0070] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0071] (i) q and r cannot both be 0; and

[0072] (ii) s, t and v cannot all be 0 simultaneously;

[0073] Z is an oligomer.

[0074] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which has the following structure:

[0075]

[0076] Wherein:

[0077] r and s are independently integers selected from 1 to 16; and

[0078] Z is an oligomer.

[0079] In another aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor used, which is formulated with an excipient and / or a carrier into a pharmaceutical composition.

[0080] In another aspect, the present invention relates to a pharmaceutical composition comprising an inhibitor according to the present invention and a pharmaceutically acceptable excipient or carrier.

[0081] In another aspect, the present invention relates to a pharmaceutical composition for treating diabetes, which comprises an inhibitor according to the present invention and a pharmaceutically acceptable excipient or carrier.

[0082] In another aspect, the present invention relates to the use of B4GALT1 as a target in identifying one or more therapeutic agents for treating diabetes.

[0083] On the other hand, the present invention relates to a method for treating or preventing diabetes, which comprises administering to a patient an inhibitor of post-translational glycosylation such as a B4GALT1 inhibitor, for example an inhibitor as defined according to the present invention.

[0084] On the other hand, the present invention relates to B4GALT1 as a biomarker for diabetes.

[0085] On the other hand, the present invention relates to the use of B4GALT1 in an in vivo method for predicting diabetes susceptibility, typically by monitoring the sequence and / or expression level and / or function of B4GALT1 in a sample obtained from a patient.

[0086] On the other hand, the present invention relates to a method for predicting diabetes susceptibility and optionally treating diabetes in a patient, the method comprising:

[0087] (a) obtaining a sample from the patient,

[0088] (b) detecting the sequence and / or expression and / or function of B4GALT1 in the sample obtained from the patient,

[0089] (c) predicting diabetes susceptibility based on the sequence and / or expression and / or function of B4GALT1 in the sample obtained from the patient,

[0090] (d) preferably administering an effective amount of a B4GALT1 inhibitor to the diagnosed patient.

[0091] On the other hand, the present invention relates to the use of an inhibitor or composition according to the present invention in the preparation of a medicament for treating diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] FIG. 1a shows an exemplary linear structure of a conjugate.

[0093] FIG. 1b shows an exemplary branched structure of a conjugate.

[0094] Figure 2-5 Shows a preferred oligomer-linker-ligand construct of the present invention.

[0095] Figure 6 Shows the details of the formulae described in items (Sentence) 1-101 disclosed herein.

[0096] Figure 7 Shows the details of the formulae described in items (Clause) 1-56 disclosed herein.

[0097] Figure 8Shows a two-dimensional display of enriched network pathways. Each pathway is represented by a dot, and the proximity of the dots is a measure of pathway similarity. Pathways with common proteins and / or adjacent ones cluster more closely together - they aggregate into higher-order processes. The "network" relationships between pathways are used to identify common biological themes. This provides a basis for further analysis to create a focused network model of key biology.

[0098] Figure 9 Is a summary figure. The analysis conducted by the mata analysis authors is shown at the top, and the further analysis conducted by the inventors is shown at the bottom. The 9 "seed" sets used for network construction on the right are from the categories of gene sets at the top.

[0099] Figure 10 Shows the improved sensitivity of the network-aware method in identifying relevant biological processes - the data analyzed using the inventors' method shows across 3 different network construction techniques - the inventors were able to address known processes in type 2 diabetes risk. Similar analysis yielded new risk-related glycosylation processes of interest to the inventors.

[0100] Figure 11 Is a schematic diagram of the network model constructed by 3 key proteins emphasized by the inventors' analysis.

[0101] Figure 12 Shows the selection of active GalNAc-siRNAs with an EC50 value less than 100 nM. After incubating with GalNAc-siRNAs targeting mouse B4GALT1 at 1000 nM in 10 consecutive dilutions for 48 hours, the dose-response of B4GALT1 gene knockdown in primary mouse hepatocytes was measured. The data was fitted to a 4-parameter sigmoidal dose-response (variable slope) equation using GraphPad Prism to determine the EC 50 value. Four active GalNAc-siRNAs, ETXM619, ETXM624, ETXM628, and ETXM633, were selected for in vivo pharmacology.

[0102] Figure 13 Is a summary of the B4GALT1 mRNA knockdown effects of multiple administrations of GalNAc-siRNAs, ETXM619, ETXM624, ETXM628, and ETXM633 (10 mg / kg) in mouse liver tissue. The y-axis values are relative mRNA expressions compared to the untreated group (n = 5). Each data point represents the relative mRNA expression of n = 3 experiments, expressed as mean ± SD. The red arrow at the top of the figure indicates the days of administration of the test article.

[0103] Figure 14Shows the effect of B4GALT1 mRNA knockdown on plasma LDL-c, glucose, and fibrinogen levels. Plasma samples were collected on day 14 after three administrations of ETXM (10 mg / kg, subcutaneous) on days 0, 3, and 7. Compared with the untreated group (n = 5), the ETXM-treated group (n = 12) showed significantly reduced LDL-c, glucose, and fibrinogen levels in normal C57BL / 6 mice. Data here are presented as mean ± SD. Detailed implementation

[0104] The present invention particularly provides inhibitors, such as oligomers like nucleic acids, such as inhibitory RNA molecules (which can be referred to as iRNA or siRNA), and compositions containing such inhibitors that can affect target expression, for example, by binding to mRNA transcribed from a gene or by inhibiting the function of nucleic acids such as long non-coding RNAs (referred to herein as "lncRNAs"). The target can be intracellular, for example, within cells of a subject (such as a human). The inhibitors can be used for preventing and / or treating medical conditions related to, for example, the expression of a target gene in a cell or the presence / activity of a nucleic acid (such as a long non-coding RNA).

[0105] Specifically, the present invention has identified post-translational glycosylation inhibitors for preventing and / or treating diabetes, such as B4GALT1 inhibitors.

[0106] B4GALT1 is β-1,4-galactosyltransferase 1, which is encoded by the B4GALT1 gene (SEQ ID NO:1) in humans.

[0107] Genomic DNA sequence (SEQ ID NO:1) containing the B4GALT1 gene

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[0151] Specifically, in the present invention, a large-scale GWAS meta-analysis (Mahajan et al., Nature Genetics, 2018, 50, p1505-1513) of 898,930 human individuals, 9% of whom had diabetes, was evaluated. Surprisingly, it was found that post-translational glycosylation was significantly associated with the risk of type 2 diabetes in normal and obese individuals. Importantly, the link between type 2 diabetes and post-translational glycosylation could not be identified by standard "functional enrichment" methods and was thus not identified by the original authors of the meta-analysis.

[0152] The computational predictions were confirmed in in vivo studies in mice (Example 9). In these studies, mice were treated with siRNA that inhibits B4GALT1 expression. In these mice, the plasma levels of LDL cholesterol, fasting glucose, and fibrinogen were significantly lower than those in untreated mice, indicating that inhibition of B4GALT1 can prevent and / or treat diabetes, particularly type 2 diabetes.

[0153] Accordingly, the present invention relates to inhibitors of targets within the post-translational glycosylation pathway, such as enzymes (e.g., B4GALT1) involved in these pathways. The inhibition can be of the gene or of the protein resulting from gene expression, and the reference to a gene (e.g., B4GALT1) herein expressly includes a reference to inhibition of gene expression or function as well as a reference to inhibition of the protein product alone.

[0154] Post-translational glycosylation preferably refers to post-translational glycosylation as seen in vivo in humans or human cells.

[0155] Definition

[0156] The "first strand" is also referred to herein as the antisense strand or the guide strand and is used interchangeably herein, and refers to a nucleic acid strand, such as a strand of siRNA (e.g., dsiRNA), which includes a region that is substantially complementary to a target sequence (e.g., to an mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence). When the complementary region is not completely complementary to the target sequence, the mismatch can be in the internal or terminal region of the molecule. In some embodiments, the double-stranded nucleic acid (e.g., the siRNA agent of the present invention) includes nucleotide mismatches in the antisense strand.

[0157] The "second strand" (also referred to herein as the sense strand or the passenger strand and used interchangeably herein) refers to a strand of nucleic acid (e.g., siRNA) that includes a region that is substantially complementary to a region of the antisense strand, as defined herein.

[0158] In the context of a molecule comprising a nucleic acid having a ligand moiety, optionally also having a linker moiety, the nucleic acid of the present invention can be referred to as an oligonucleotide moiety or an oligonucleoside moiety.

[0159] An oligonucleotide is a short nucleic acid polymer. Although oligonucleotides contain phosphodiester bonds between their nucleoside components (base plus sugar), the present invention is not limited to oligonucleotides that are always linked by such phosphodiester bonds between adjacent nucleosides, and other oligomers of nucleosides linked by bonds other than phosphodiester bonds are also contemplated. For example, the bond between nucleotides can be a phosphorothioate bond. Thus, the term "oligonucleoside" as used herein encompasses oligonucleotides and other oligomers of nucleosides. According to the present invention, it is preferred that the oligonucleoside is a nucleic acid having at least a portion that is an oligonucleotide. According to the present invention, it is also preferred that the oligonucleoside has one or more or most phosphodiester backbone bonds between nucleosides. According to the present invention, it is also preferred that the oligonucleoside has one or more or most phosphodiester backbone bonds between nucleosides and also has one or more phosphorothioate backbone bonds between nucleosides (usually in the terminal regions of the first strand and / or the second strand).

[0160] In some embodiments, the double-stranded nucleic acid (e.g., the siRNA agent) of the present invention includes nucleoside mismatches in the sense strand. In some embodiments, the nucleoside mismatch is within, for example, 5, 4, 3, 2, or 1 nucleoside from the 3' end of the nucleic acid (e.g., siRNA).

[0161] In another embodiment, the nucleoside mismatch is in the 3'-terminal nucleoside of the nucleic acid (e.g., siRNA), for example.

[0162] "Target sequence" (which may be referred to as target RNA or target mRNA) refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, including mRNA that is a product of the primary transcript of RNA processing, or may be a continuous portion of the nucleotide sequence of any RNA molecule (such as lncRNA that needs to be inhibited).

[0163] The length of the target sequence can be about 10 - 35 nucleotides, for example, about 15 - 30 nucleotides in length. For example, the length of the target sequence can be about 15 - 30, 15 - 29, 15 - 28, 15 - 27, 15 - 26, 15 - 25, 15 - 24, 15 - 23, 15 - 22, 15 - 21, 15 - 20, 15 - 19, 15 - 18, 15 - 17, 18 - 30, 18 - 29, 18 - 28, 18 - 27, 18 - 26, 18 - 25, 18 - 24, 18 - 23, 18 - 22, 18 - 21, 18 - 20, 19 - 30, 19 - 29, 19 - 28, 19 - 27, 19 - 26, 19 - 25, 19 - 24, 19 - 23, 19 - 22, 19 - 21, 19 - 20, 20 - 30, 20 - 29, 20 - 28, 20 - 27, 20 - 26, 20 - 25, 20 - 24, 20 - 23, 20 - 22, 20 - 21, 21 - 30, 21 - 29, 21 - 28, 21 - 27, 21 - 26, 21 - 25, 21 - 24, 21 - 23 or 21 - 22 nucleotides. Ranges and lengths between the above ranges and lengths are also considered to be part of the present invention.

[0164] The term "ribonucleoside" or "nucleotide" may also refer to a modified nucleotide, as further detailed below.

[0165] The nucleic acid can be DNA or RNA, and can contain modified nucleotides. RNA is the preferred nucleic acid.

[0166] The terms "iRNA", "siRNA", "RNAi agent" and "iRNA agent", "RNA interference agent", which are used interchangeably herein, refer to an RNA-containing reagent that mediates the targeted cleavage of an RNA transcript through the RNA-induced silencing complex (RISC) pathway. siRNA directs the sequence-specific degradation of mRNA through RNA interference (RNAi).

[0167] Double-stranded RNA is referred to herein as "double-stranded siRNA (dsiRNA) agent", "double-stranded siRNA (dsiRNA) molecule", "double-stranded RNA (dsRNA) agent", "double-stranded RNA (dsRNA) molecule", "dsiRNA agent", "dsiRNA molecule", or "dsiRNA", which refers to a complex of ribonucleic acid molecules having a duplex comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target RNA. Most of the nucleosides of each strand of the nucleic acid (such as a dsiRNA molecule) are preferably ribonucleosides, but in such cases, each strand or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides or modified ribonucleosides. In addition, as used in this specification, "siRNA" may include chemically modified ribonucleosides.

[0168] The term "modified nucleoside" refers to a nucleoside independently having a modified sugar moiety, a modified internucleoside bond, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleoside" encompasses substitutions, additions, or removals of internucleoside bonds, sugar moieties, or nucleobases (such as functional groups or atoms). For the purposes of this specification and the claims, any such modifications (as used in siRNA-type molecules) are included within "iRNA" or "RNAi agent" or "siRNA" or "siRNA agent".

[0169] The length of the duplex region of the nucleic acid of the present invention, such as dsRNA, can be about 9 to 40 base pairs, for example, 9 to 36 base pairs in length, for example, about 15 - 30 base pairs in length, for example, 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, for example, about 15 - 30, 15 - 29, 15 - 28, 15 - 27, 15 - 26, 15 - 25, 15 - 24, 15 - 23, 15 - 22, 15 - 21, 15 - 20, 15 - 19, 15 - 18, 15 - 17, 18 - 30, 18 - 29, 18 - 28, 18 - 27, 18 - 26, 18 - 25, 18 - 24, 18 - 23, 18 - 22, 18 - 21, 18 - 20, 19 - 30, 19 - 29, 19 - 28, 19 - 27, 19 - 26, 19 - 25, 19 - 24, 19 - 23, 19 - 22, 19 - 21, 19 - 20, 20 - 30, 20 - 29, 20 - 28, 20 - 27, 20 - 26, 20 - 25, 20 - 24, 20 - 23, 20 - 22, 20 - 21, 21 - 30, 21 - 29, 21 - 28, 21 - 27, 21 - 26, 21 - 25, 21 - 24, 21 - 23 or 21 - 22 base pairs in length.

[0170] The two strands forming the duplex structure can be different parts of a larger molecule, or they can be separate molecules, such as RNA molecules.

[0171] The term "nucleotide overhang" refers to at least one unpaired nucleotide extending from the duplex structure of a double-stranded nucleic acid. The ds nucleic acid can contain at least one nucleotide overhang; alternatively, the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more nucleotides. The nucleotide overhang can contain or consist of nucleotide analogs (including deoxynucleotides). The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof.

[0172] In addition, the nucleotide(s) of the overhang can be present at the 5'-end, 3'-end or both ends of the antisense strand or the sense strand.

[0173] In certain embodiments, the antisense strand has 1 - 10 nucleotides at the 3'-end or 5'-end, for example, 0 - 3, 1 - 3, 2 - 4, 2 - 5, 4 - 10, 5 - 10, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide overhangs.

[0174] "Flat" or "blunt end" means that there are no unpaired nucleosides at this end of the double-stranded nucleic acid, i.e., no nucleoside overhangs. The nucleic acids of the present invention include nucleic acids having no nucleoside overhangs at one end or no nucleoside overhangs at either end.

[0175] As understood by those skilled in the art, unless otherwise specified, the term "complementary", when used to describe a first nucleoside sequence relative to a second nucleoside sequence, refers to the ability of an oligonucleoside containing the first nucleoside sequence to hybridize with an oligonucleoside or polynucleoside containing the second nucleoside sequence and form a duplex structure under certain conditions. Such conditions can be, for example, stringent conditions, which can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C for 12 - 16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press).

[0176] As described herein, complementary sequences within a nucleic acid (e.g., dsiRNA) include base pairing of an oligonucleoside or polynucleoside containing a first nucleoside sequence with an oligonucleoside or polynucleoside containing a second nucleoside sequence over the entire length of one or both nucleoside sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" or "partially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, but preferably no more than 5, while maintaining the ability to hybridize under conditions most relevant to their ultimate application (e.g., inhibiting gene expression via the RISC pathway). In the determination of complementarity, overhangs should not be considered mismatches. For example, a nucleic acid (e.g., dsRNA) containing an oligonucleoside of 17 nucleosides in length and another oligonucleoside of 19 nucleosides in length, where the longer oligonucleoside contains a sequence of 17 nucleosides that is fully complementary to the shorter oligonucleoside, can still be referred to as "fully complementary".

[0177] As used herein, "complementary" sequences may also include non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, or consisting entirely of non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, provided that the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairing.

[0178] The terms "complementary", "fully complementary", and "substantially / partially complementary" as used herein can be used to refer to base pairing 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.

[0179] In 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 are partially complementary if they form a duplex region that is at least 17 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0180] In certain embodiments, the first and second strands of the nucleic acid according to the present invention are partially complementary if they form a duplex region that is 19 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs. In certain embodiments, the first and second strands of the nucleic acid according to the present invention are partially complementary if they form a duplex region that is 21 base pairs in length and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0181] Alternatively, the first and second strands of the nucleic acid according to the present invention are partially complementary if they form a duplex region that is at least 17 base pairs in length, wherein at least 14, 15, 16, or 17 of the base pairs are complementary base pairs, particularly Watson-Crick base pairs.

[0182] In certain embodiments, the first and second strands of the nucleic acid according to the present invention are partially complementary if they form a duplex region that is 19 base pairs in length, wherein at least 14, 15, 16, 17, 18, or all 19 base pairs are complementary base pairs, particularly Watson-Crick base pairs. In certain embodiments, the first and second strands of the nucleic acid according to the present invention are partially complementary if they form a duplex region that is 21 base pairs in length, wherein at least 16, 17, 18, 19, 20, or all 21 base pairs are complementary base pairs, particularly Watson-Crick base pairs.

[0183] As used herein, a nucleic acid that is "substantially complementary" or "partially complementary" to at least a portion of a messenger RNA (mRNA) refers to a polynucleoside that is substantially or partially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding a gene). In certain embodiments, the continuous portion of the mRNA is a sequence listed in Table 1, i.e., any one of SEQ ID NOs: 2-21 or 102-201. For example, a polynucleoside is complementary to at least a portion of the mRNA of a gene of interest if the sequence is substantially or partially complementary to an uninterrupted portion of the mRNA encoding the gene of interest.

[0184] Thus, in some preferred embodiments, the antisense oligonucleotides disclosed herein are fully complementary to the target gene sequence.

[0185] In other embodiments, the antisense oligonucleotides disclosed herein are substantially or partially complementary to the target RNA sequence and comprise a continuous nucleoside sequence that is at least about 80% complementary to the equivalent region of the target RNA sequence over its entire length, such as at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary or 100% complementary.

[0186] In certain embodiments, the first strand (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 the 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 the 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 (i.e., any one of SEQ ID NO: 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 NO: 102-201.

[0187] In certain embodiments, if the first strand (antisense strand) of a nucleic acid according to the invention comprises a continuous nucleoside sequence of at least 17 nucleosides, it is partially complementary to a continuous portion of B4GALT1 mRNA, wherein at least 14, 15, 16 or 17 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of B4GALT1 mRNA. In certain embodiments, the first strand of a nucleic acid according to the invention comprises a continuous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NO: 2-21 or 102-201). In certain embodiments, the first strand of a nucleic acid according to the invention comprises a continuous nucleoside sequence of 19 nucleosides, wherein at least 14, 15, 16, 17, 18 or all 19 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1 (i.e., any one of SEQ ID NO: 2-21 or 102-201). In certain embodiments, the first strand of a nucleic acid according to the invention comprises a continuous nucleoside sequence of 21 nucleosides, wherein at least 16, 17, 18, 19, 20 or all 21 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of SEQ ID NO: 102-201. In certain embodiments, the first strand of a nucleic acid according to the invention comprises a continuous nucleoside sequence of 23 nucleosides, wherein at least 18, 19, 20, 21, 22 or all 23 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of SEQ ID NO: 102-201.

[0188] In some embodiments, a nucleic acid of the invention (e.g., siRNA) comprises a sense strand that is substantially or partially complementary to an antisense polynucleotide, which in turn is complementary to a target gene sequence and comprises a continuous nucleoside sequence that is at least about 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary, to an equivalent region of the nucleoside sequence of the antisense strand over its entire length.

[0189] In some embodiments, a nucleic acid of the invention (e.g., siRNA) comprises an antisense strand that is substantially or partially complementary to a target sequence and comprises a continuous nucleoside sequence that is at least 80% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary, to the target sequence over its entire length.

[0190] As used herein, "subject" refers to an animal such as a mammal, including a primate (e.g., human, non-human primate such as monkey and chimpanzee) or non-primate or bird that expresses an endogenous or heterologous target gene when the target gene sequence has sufficient complementarity to a nucleic acid (e.g., an iRNA agent) to facilitate target gene knockdown. In certain preferred embodiments, the subject is human.

[0191] The terms "treating / treatment" refer to a beneficial or desired result, including but not limited to alleviating or ameliorating one or more symptoms associated with gene expression. "Treatment" can also mean an extended survival rate compared to the expected survival rate without treatment. Treatment can include preventing the development of comorbidities, such as reducing liver damage in a subject with a liver infection.

[0192] As used herein, "therapeutically effective amount" is intended to include an amount of a nucleic acid (e.g., iRNA) that, when administered to a patient to treat a subject having a disease, is sufficient to effect treatment of the disease (e.g., by attenuating, ameliorating or maintaining one or more symptoms of an existing disease or disorder or its associated comorbidities).

[0193] The phrase "pharmaceutically acceptable" as used herein refers to a compound, material, composition or dosage form that is suitable for use in contact with the tissues of human and animal subjects without undue toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0194] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not injurious to the subject being treated.

[0195] When listing values of parameters or ranges of values, it is intended that values or ranges intermediate to the listed values also be part of the present invention.

[0196] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article.

[0197] The term "comprising" is used herein to mean the phrase "comprising but not limited to" and can be used interchangeably with that phrase.

[0198] The term "or" as used herein refers to the term and, unless the context clearly indicates otherwise, is interchangeable with the term "and / or". For example, "sense strand or antisense strand" should be understood to mean "sense strand or antisense strand or sense strand and antisense strand".

[0199] The term "about" as used herein is used to denote within the typical tolerances in the art. For example, "about" can be understood to be about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When "about" appears before a series of numbers or a range, it should be understood that "about" can modify each number in the series or range.

[0200] The term "at least" before a number or a series of numbers should be understood to include the number adjacent to the term "at least", as well as all subsequent numbers or integers that can be logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a 21 - nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0201] As used herein, "not greater than" or "less than" should be understood to include the value adjacent to the phrase and the logically lower value or integer, as can be logically derived from the context, and should be understood to include the value adjacent to zero. For example, a duplex having an overhang "not greater than 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "not greater than" appears before a series of numbers or a range, it should be understood that "not greater than" can modify each number in the series or range.

[0202] The terminal region of a strand is the last 5 nucleotides from the 5' or 3' end.

[0203] The nucleobase sequence is the base sequence of the nucleic acid in an oligomer.

[0204] One of ordinary skill in the art can appropriately combine the various embodiments of the present invention.

[0205] Target

[0206] The targets for inhibition disclosed herein can be, but are not limited to, mRNA, lncRNA, polypeptide, protein, or gene.

[0207] The targets herein are targets involved in the protein post - translational glycosylation pathway. These are preferred targets, and inhibition of these targets helps in the prevention or treatment of diabetes. A preferred target for inhibition is B4GALT1, and inhibition can be effected by inhibiting the expression or function of the gene or the protein or both.

[0208] In one aspect, the target is mRNA expressed by a gene or long non-coding RNA (lncRNA).

[0209] In a preferred embodiment, the target is mRNA produced by expression of the B4GALT1 gene. Exemplary target sequences on B4GALT1 mRNA are listed in Table 1 below.

[0210] Table 1

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] It should be understood that SEQ ID NO: 2 to 21 and 102 to 201 relate to human (Homo sapiens) mRNA sequences.

[0217] Disease / Condition

[0218] The present invention relates to inhibitors suitable for use in or for the treatment of diabetes (such as type 1 or type 2 diabetes, preferably type 2 diabetes).

[0219] Inhibitor

[0220] The inhibitors of the present invention include nucleic acids such as siRNA, antibodies and antigen-binding fragments thereof, such as monoclonal antibodies, polypeptides, antibody-drug conjugates and small molecules. Preferably, the nucleic acid is, for example, siRNA.

[0221] Certain preferred features of the inhibitors of the present invention are given below, wherein these are oligonucleosides, such as siRNA.

[0222] In certain embodiments, the nucleic acid comprises a first strand that comprises a sequence that is 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 that comprises a sequence that is at least partially complementary to B4GALT1 mRNA (NM_001497.4).

[0223] In certain embodiments, the nucleic acid for inhibiting B4GALT1 expression comprises a duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is:

[0224] (i) at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, and

[0225] (ii) comprises at least 17 consecutive nucleotides that differ from any of SEQ ID NOs: 22 - 41 or 202 - 301 by 0 or 1 nucleotide.

[0226] In certain embodiments, the first strand comprises nucleotides 2 - 18 of any of the sequences shown in SEQ ID NOs: 22 - 41 or 202 - 301.

[0227] In certain embodiments, the nucleic acid for inhibiting B4GALT1 expression comprises a duplex region that includes a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is:

[0228] (i) at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, and

[0229] (ii) comprises at least 21 consecutive nucleotides that differ from any of SEQ ID NOs: 202 - 301 by 0 or 1 nucleotide.

[0230] In certain embodiments, the first strand comprises nucleotides 2 - 22 of any of the sequences shown in SEQ ID NOs: 202 - 301.

[0231] In certain embodiments, the first strand comprises any of SEQ ID NOs: 22 - 41 or 202 - 301.

[0232] In certain embodiments, the second strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differ from any of SEQ ID NOs: 42 - 61 or 302 - 401 by 0 or 1 nucleotide; wherein the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleotides.

[0233] In certain embodiments, the second strand comprises a nucleotide sequence of at least 19 consecutive nucleotides that differ from any of SEQ ID NOs: 302 - 401 by 0 or 1 nucleotide; wherein the second strand has a region that is at least 85% complementary to the first strand over 19 consecutive nucleotides.

[0234] In certain embodiments, the second strand comprises a nucleotide sequence of at least 21 consecutive nucleotides that differ from any of SEQ ID NOs: 302 - 401 by 0 or 1 nucleotide; wherein the second strand has a region that is at least 85% complementary to the first strand over 21 consecutive nucleotides.

[0235] In certain embodiments, the second strand comprises any of SEQ ID NOs: 42 - 61 or 302 - 401.

[0236] In certain embodiments, the nucleic acid comprises a first strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any of SEQ ID NOs: 22-41 or 202-301 by 0 or 1 nucleoside; and a second strand comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any of SEQ ID NOs: 42-61 or 302-401 by 0 or 1 nucleoside.

[0237] Preferably herein, a duplex region is formed between the first strand (antisense strand) and the complementary second strand (sense). Table 2 below lists exemplary pairs of complementary antisense and sense strands:

[0238] Table 2

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248] In certain embodiments, the invention relates to a nucleic acid comprising a first strand and a second strand, the first and second strands comprising, consisting of, or consisting essentially of a nucleoside sequence that differs from any of the following first and second sequences by 0 or 1 nucleoside:

[0249] Unmodified First Strand Unmodified Second Strand SEQ ID NO:202 SEQ ID NO:302 SEQ ID NO:205 SEQ ID NO:305 SEQ ID NO:217 SEQ ID NO:317 SEQ ID NO:228 SEQ ID NO:328

[0250] In certain embodiments, the nucleic acid for inhibiting B4GALT1 expression comprises a duplex region comprising a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is:

[0251] (i) at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, and

[0252] (ii) comprises at least 17 consecutive nucleosides that differ from any of SEQ ID NOs: 62-81 or 402-513 by 0 or 1 nucleoside.

[0253] In certain embodiments, the first strand comprises nucleotides 2-18 of any of the sequences shown in SEQ ID NOs: 62-81 or 402-513.

[0254] In certain embodiments, the nucleic acid for inhibiting B4GALT1 expression comprises a duplex region that includes a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is:

[0255] (i) at least partially complementary to a portion of the RNA transcribed from the B4GALT1 gene, and

[0256] (ii) comprises at least 21 consecutive nucleotides that differ from any of SEQ ID NOs: 402-513 by 0 or 1 nucleotide.

[0257] In certain embodiments, the first strand comprises nucleotides 2-22 of any of the sequences shown in SEQ ID NOs: 402-513.

[0258] In certain embodiments, the first strand comprises any of SEQ ID NOs: 62-81 or 402-513.

[0259] The modification patterns of the nucleic acids shown in SEQ ID NOs: 62-81 and 402-513 are summarized in Table 3 below:

[0260] Table 3

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271] In certain embodiments, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs from any of SEQ ID NOs: 82-101 or 514-621 by 0 or 1 nucleoside; wherein the second strand has a region that is at least 85% complementary to the first strand over the 17 consecutive nucleosides.

[0272] In certain embodiments, the second strand comprises a nucleoside sequence of at least 19 consecutive nucleosides that differs from any of SEQ ID NOs: 514-621 by 0 or 1 nucleoside; wherein the second strand has a region that is at least 85% complementary to the first strand over the 19 consecutive nucleosides.

[0273] In certain embodiments, the second strand comprises a nucleoside sequence of at least 21 consecutive nucleosides that differs from any of SEQ ID NOs: 514-621 by 0 or 1 nucleoside; wherein the second strand has a region that is at least 85% complementary to the first strand over the 21 consecutive nucleosides.

[0274] In certain embodiments, the second strand comprises any of SEQ ID NOs: 82-101 or 514-621.

[0275] The modification patterns of the nucleic acids shown in SEQ ID NOs: 82-101 and 514-621 are summarized in Table 4 below:

[0276] Table 4

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] As used herein, and particularly in Tables 3 and 4, the following abbreviations are used to denote modified nucleosides:

[0288] Am represents 2'-O-methyladenosine, Cm represents 2'-O-methylcytidine, Gm represents 2'-O-methylguanosine, Um represents 2'-O-methyluridine, Af represents 2'-fluoro-adenosine, Cf represents 2'-fluoro-cytidine, Gf represents 2'-fluoro-guanosine, and Uf represents 2'-fluoro-uridine.

[0289] In addition, the letter "s" is used as an abbreviation for a phosphorothioate bond between two consecutive (modified) nucleosides. For example, the abbreviation "AmsAm" is used for two consecutive 2'-O-methyladenosine nucleosides linked by a 3'-5' phosphorothioate bond. Nucleosides linked by a standard 3'-5' phosphodiester bond do not use an abbreviation. For example, the abbreviation "AmAm" is used for two consecutive 2'-O-methyladenosine nucleosides linked by a 3'-5' phosphodiester bond.

[0290] In certain embodiments, the nucleic acid comprises a first strand that comprises, consists of, or consists essentially of 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 that comprises, consists of, or consists essentially of a (modified) nucleoside sequence that differs from any one of SEQ ID NOs: 82-101 or 514-621 by 0 or 1 nucleoside.

[0291] Preferred combinations of complementary modified antisense strands (first strands) and sense strands (second strands) are listed in Table 5 below:

[0292] Table 5

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] In a particularly preferred embodiment, the invention relates to a nucleic acid comprising a first strand and a second strand, wherein the first strand and the second strand comprise, consist of, or consist essentially of a nucleoside sequence that differs from any one of the following first sequence and second sequence by 0 or 1 nucleoside:

[0300] Modified First Strand Modified Second Strand SEQ ID NO:502(ETXS2400) SEQ ID NO:610(ETXS2399) SEQ ID NO:503(ETXS2402) SEQ ID NO:611(ETXS2401) SEQ ID NO:504(ETXS2406) SEQ ID NO:612(ETXS2405) SEQ ID NO:505(ETXS2408) SEQ ID NO:613(ETXS2407) SEQ ID NO:506(ETXS2424) SEQ ID NO:614(ETXS2423) SEQ ID NO:507(ETXS2426) SEQ ID NO:615(ETXS2425) SEQ ID NO:508(ETXS2430) SEQ ID NO:616(ETXS2429) SEQ ID NO:509(ETXS2432) SEQ ID NO:617(ETXS2431) SEQ ID NO:510(ETXS2434) SEQ ID NO:611(ETXS2401) SEQ ID NO:511(ETXS2436) SEQ ID NO:613(ETXS2407) SEQ ID NO:512(ETXS2438) SEQ ID NO:615(ETXS2425) SEQ ID NO:513(ETXS2440) SEQ ID NO:617(ETXS2431)

[0301] In the event of any ambiguity between the sequences in this specification and those in the accompanying sequence listing, the sequences provided herein are considered to be the correct sequences.

[0302] Abasic Nucleotide

[0303] In certain embodiments, the nucleic acid according to the invention contains 1, for example 2, for example 3, for example 4 or more abasic nucleosides. Abasic nucleosides are modified nucleosides as they lack the base normally seen at position 1 of the sugar moiety. Generally, according to the invention, there is a hydrogen at position 1 of the sugar moiety of the abasic nucleoside present in the nucleic acid.

[0304] The abasic nucleosides are in the terminal region of the second strand, preferably within the terminal 5 nucleosides at the end of the strand. The terminal region can be the terminal 5 nucleosides, which includes the abasic nucleoside.

[0305] As a preferred feature, the second strand can contain (all are specifically considered for combination unless mutually exclusive):

[0306] 2 or more abasic nucleosides in the terminal region of the second strand; and / or

[0307] 2 or more abasic nucleosides in the 5' or 3' terminal region of the second strand; and / or

[0308] 2 or more abasic nucleosides in the 5' or 3' terminal region of the second strand, where the abasic nucleosides are present in the overhangs as described herein; and / or

[0309] 2 or more consecutive abasic nucleosides in the terminal region of the second strand, where preferably one of such abasic nucleosides is the terminal nucleoside; and / or

[0310] 2 or more consecutive abasic nucleosides in the 5' or 3' terminal region of the second strand, where preferably one of such abasic nucleosides is the terminal nucleoside which is the terminal nucleoside of the 5' or 3' terminal region of the second strand; and / or

[0311] Reverse internucleoside bonds link at least one abasic nucleoside to an adjacent base nucleoside in the terminal region of the second strand; and / or

[0312] Reverse internucleoside bonds link at least one abasic nucleoside to an adjacent base nucleoside in the 5' or 3' terminal region of the second strand; and / or

[0313] An abasic nucleoside as the penultimate nucleoside, which is linked by a reverse bond to a nucleoside that is not the terminal nucleoside (referred to herein as the antepenultimate nucleoside); and / or

[0314] When reading the strand in the direction towards the end containing the terminal nucleoside, the abasic nucleoside serves as two terminal nucleosides linked by a 5'-3' bond;

[0315] When reading the strand in the direction towards the end containing the terminal nucleoside, the abasic nucleoside serves as two terminal nucleosides linked by a 3'-5' bond;

[0316] The abasic nucleoside serves as the two terminal positions, where the penultimate nucleoside is linked to the antepenultimate nucleoside by a reverse bond, and where the reverse bond is a 5-5' reverse bond or a 3'-3' reverse bond;

[0317] The abasic nucleoside serves as the two terminal positions, where the penultimate nucleoside is linked to the antepenultimate nucleoside by a reverse bond, and where

[0318] (1) the reverse bond is a 5-5' reverse bond, and when reading towards the end containing the terminal abasic nucleoside and the penultimate abasic nucleoside, the bond between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'5'; or

[0319] (2) the reverse bond is a 3-3' reverse bond, and when reading towards the end containing the terminal abasic nucleoside and the penultimate abasic nucleoside, the bond between the terminal abasic nucleoside and the penultimate abasic nucleoside is 5'3'.

[0320] Preferably, there is an abasic nucleoside at the end of the second strand.

[0321] Preferably, in the terminal region of the second strand, preferably there are two or at least two abasic nucleosides at the end and the penultimate position.

[0322] Preferably, two or more abasic nucleosides are consecutive. For example, all abasic nucleosides can be consecutive. For example, the terminal 1 or terminal 2 or terminal 3 or terminal 4 nucleosides can be abasic nucleosides.

[0323] An abasic nucleoside can also be linked to an adjacent nucleoside by a 5'-3' phosphodiester bond or a reverse bond, unless there is only one abasic nucleoside at the end, in which case it will have a reverse bond with the adjacent nucleoside.

[0324] The reverse bond (which can also be called an inverted bond and is also visible in the art) includes a 5'-5', 3'-3', 3'-2' or 2'-3' phosphodiester bond between adjacent sugar moieties of the nucleosides.

[0325] An abasic nucleoside that is not at the end will have two phosphodiester bonds, one for each adjacent nucleoside, and these bonds can be reverse bonds, or can be 5'-3 phosphodiester bonds, or can each be one.

[0326] Preferred embodiments include 2 abasic nucleosides at the end and the penultimate position of the second strand, and wherein the reverse internucleoside bond is between the penultimate (abasic) nucleoside and the antepenultimate nucleoside.

[0327] Preferably, there are 2 abasic nucleosides at the end and the penultimate position of the second strand, and the penultimate nucleoside is linked to the antepenultimate nucleoside by a reverse internucleoside bond and to the terminal nucleoside by a 5'-3' or 3'-5' phosphodiester bond (read in the direction of the end of the molecule).

[0328] Different preferred features are as follows:

[0329] The reverse internucleoside bond is a 3'-3' reverse bond. The reverse internucleoside bond is located in the terminal region away from the 5'-end phosphate of the second strand.

[0330] The reverse internucleoside bond is a 5'-5' reverse bond. The reverse internucleoside bond is located in the terminal region away from the 3'-end hydroxyl of the second strand.

[0331] In certain embodiments, the second strand contains 2 consecutive abasic nucleosides in the 5'-terminal region of the second strand, where one of these abasic nucleosides is the terminal nucleoside of the 5'-terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside of the 5'-terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the adjacent 5'-proximal region adjacent first base nucleoside by a reverse internucleoside bond; (b) the reverse bond is a 5-5' reverse bond; and (c) when reading towards the end containing the terminal abasic nucleoside and the penultimate abasic nucleoside, the bond between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3'-5'. More typically, (i) the first and second strands each have 19 or 23 nucleosides in length; (ii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 5'-proximal region of the second strand, where the first phosphorothioate internucleoside bond is between the adjacent first base nucleoside of (a) and the adjacent second base nucleoside in the 5'-proximal region of the second strand, and the second phosphorothioate internucleoside bond is between the adjacent second base nucleoside and the adjacent third base nucleoside in the 5'-proximal region of the second strand; (iii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 5'- and 3'-terminal regions of the first strand, whereby the terminal nucleosides at each of the 5'- and 3'-terminal regions of the first strand are respectively linked to their respective 5'- and 3'-adjacent penultimate nucleosides by phosphorothioate internucleoside bonds, and the first 5'- and 3'-penultimate nucleosides are each linked to their respective 5'- and 3'-adjacent antepenultimate nucleosides by phosphorothioate internucleoside bonds; 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.

[0332] Alternatively, the second strand comprises two consecutive abasic nucleosides, preferably in the overhang in the 3'-terminal region of the second strand, where one of the abasic nucleosides is the terminal nucleoside in the 3'-terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside in the 3'-terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the first base nucleoside adjacent to the adjacent 3'-proximal terminal region by a reverse internucleoside bond; (b) the reverse bond is a 3-3' reverse bond; and (c) when reading towards the end comprising the terminal abasic nucleoside and the penultimate abasic nucleoside, the bond between the terminal abasic nucleoside and the penultimate abasic nucleoside is 5'-3'. More typically, (i) each of the first strand and the second strand has a length of 19 or 23 nucleosides; (ii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 3'-proximal terminal region of the second strand, wherein the first phosphorothioate internucleoside bond is between the adjacent first base nucleoside in (a) and the adjacent second base nucleoside in the 3'-proximal terminal region of the second strand, and the second phosphorothioate internucleoside bond is between the adjacent second base nucleoside and the adjacent third base nucleoside in the 3'-proximal terminal region of the second strand; (iii) two phosphorothioate internucleoside bonds are respectively between three consecutive positions in the 5'- and 3'-terminal regions of the first strand, whereby the terminal nucleosides at each of the 5'- and 3'-terminal regions of the first strand are respectively linked to their respective 5'- and 3'-adjacent penultimate nucleosides by phosphorothioate internucleoside bonds, and the first 5'- and 3'-penultimate nucleosides are respectively linked to their respective 5'- and 3'-adjacent penultimate nucleosides by phosphorothioate internucleoside bonds; 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.

[0333] Structural examples are as follows (wherein the specific RNA nucleosides shown are not limiting and can be any RNA nucleoside):

[0334] A 3-3' reverse bond (and also shows the 5'-3' direction of the last phosphodiester bond between two abasic molecules when reading towards the end of the molecule)

[0335]

[0336] B shows a 5'-5' reverse bond (and also shows the 3'-5' direction of the last phosphodiester bond between two abasic molecules when reading towards the end of the molecule)

[0337]

[0338] Providing an abasic nucleoside or abasic nucleosides present in a nucleic acid in the presence of a reverse internucleoside bond or bonds (i.e., 5'-5' or 3'-3' reverse internucleoside bonds). The reverse bond occurs due to a change in the orientation of adjacent nucleoside sugars such that the sugar has a 3'–5' orientation, opposite to the conventional 5'–3' orientation (referring to the numbering of the ring atoms on the nucleoside sugar). One or more abasic nucleosides present in the nucleic acids of the present invention preferably include such inverted nucleoside sugars.

[0339] In the case where the terminal nucleoside has an inverted orientation, this will result in an "inverted" end configuration of the entire nucleic acid. Although some of the structures depicted and referred to herein are represented using the conventional 5'–3' direction (referring to the numbering of the ring atoms on the nucleoside sugar), it will be understood that the presence of a terminal nucleoside with an orientation change and a proximal 3'-3' reverse bond will result in the nucleic acid having an overall 5'-5' end structure (i.e., the conventional 3' terminal nucleoside becomes the 5' terminal nucleoside). Alternatively, it will be understood that the presence of a terminal nucleoside with an orientation change and a proximal 5'-5' reverse bond will result in the nucleic acid having an overall 3'-3' end structure.

[0340] The proximal 3'-3' or 5'-5' reverse bonds as described herein can include a reverse bond directly adjacent / linked to a terminal nucleoside with an inverted orientation, such as a single terminal nucleoside with an inverted orientation. Alternatively, the proximal 3'-3' or 5'-5' reverse bonds as described herein can include reverse bonds of two or more adjacent nucleosides with an inverted orientation, such as two or more terminal region nucleosides with an inverted orientation, such as a terminal nucleoside and the penultimate nucleoside. In this way, the reverse bond can be linked to the penultimate nucleoside with an inverted orientation. Although those skilled in the art will understand that the inverted orientation as described above can result in the nucleic acid molecule having an overall 3'-3' or 5'-5' end structure as described herein, it should also be understood that due to the presence of one or more additional reverse bonds and / or nucleosides with an inverted orientation, the overall nucleic acid can have a 3'-5' end structure corresponding to the conventionally positioned 5' / 3' ends.

[0341] In one aspect, the nucleic acid can have a 3'-3' reverse bond, and the terminal sugar moiety can contain a 5'OH at the 5' position of the terminal sugar, rather than a 5' phosphate group.

[0342] Thus, those skilled in the art will clearly understand that the 5'-5', 3'-3', and 3'-5' (read along that terminal direction) end variants of the more conventional 5'-3' structures (referring to the ring atom numbering on the terminal nucleoside sugar) depicted herein are included within the scope of the present disclosure, where there is one reverse bond or more reverse bonds.

[0343] For example, in the case where there is an inverted internucleoside bond and / or one or more nucleosides having an inverted orientation resulting in inverted termini, and the relative position of the bond (e.g., relative position to the linker) or the position of internal features (e.g., modified nucleosides) is defined relative to the 5' or 3' end of the nucleic acid, then the 5' or 3' end is the conventional 5' or 3' end that would be present if there were no inverted linkages, and wherein the conventional 5' and 3' ends are determined by considering the directionality of the majority of the internal nucleoside bonds and / or the orientation of the nucleosides within the nucleic acid. From these internal bonds and / or nucleoside orientations, it can be determined which termini of the nucleic acid would constitute the conventional 5' and 3' ends of the molecule without inverted bonds (with reference to the ring atom numbering on the terminal nucleoside sugar).

[0344] For example, in the structure shown below, there are no base residues at the first 2 positions located at the "5'" end. When the terminal nucleoside has an inverted orientation, the "5'" end shown in the figure below (which is the conventional 5' end) can actually contain a 3' OH according to the inverted nucleoside at the terminal position. Nevertheless, when read in the standard 5'[PO4] to 3'[OH] direction of the nucleic acid molecule (with reference to the ring atom numbering on the nucleoside sugar), most of the molecule will contain conventional internucleoside bonds from the 3' OH of the sugar to the 5' phosphate of the next sugar, which can be used to determine the conventional 5' and 3' ends found in the absence of an inverted terminus configuration.

[0345] A 5’A-A-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me 3’

[0346] The inverted bond is preferably located at the end of the nucleic acid, such as at the end of an RNA that is away from the ligand portion of the molecule (e.g., the portion containing GalNAc).

[0347] A GalNAc-siRNA construct having 5'-GalNAc on the sense strand can have an inverted bond at the other end of the sense strand.

[0348] A GalNAc-siRNA construct having 3'-GalNAc on the sense strand can have an inverted bond at the other end of the sense strand.

[0349] Nucleic Acid Length

[0350] In one aspect, i) the length of the first strand of the nucleic acid ranges from 17 to 30 nucleosides, preferably from 19 to 25 nucleosides, more preferably 19 or 23 nucleosides; and / or ii) the length of the second strand of the nucleic acid ranges from 17 to 30 nucleosides, preferably from 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0351] Typically, the length of the double-stranded region of the nucleic acid is 17 to 30 nucleotides, more preferably 19 or 21 nucleotides. Similarly, the length of the complementary region between the first strand and the RNA portion transcribed from the B4GALT1 gene is 17 to 30 nucleotides.

[0352] In one aspect, i) the length of the first strand of the nucleic acid ranges from 15 to 30 nucleotides, preferably from 19 to 25 nucleotides, more preferably 23 or 25 nucleotides; and / or

[0353] ii) the length of the second strand of the nucleic acid ranges from 15 to 30 nucleotides, preferably from 19 to 25 nucleotides, more preferably 23 nucleotides.

[0354] Typically, the length of the double-stranded structure of a nucleic acid such as iRNA is about 15 to 30 base pairs, such as 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present invention.

[0355] Similarly, the length of the complementary region of the antisense sequence to the target sequence and / or the complementary region of the antisense sequence to the sense sequence is about 15 to 30 nucleotides, such as 15 - 29, 15 - 28, 15 - 27, 15 - 26, 15 - 25, 15 - 24, 15 - 23, 15 - 22, 15 - 21, 15 - 20, 15 - 19, 15 - 18, 15 - 17, 18 - 30, 18 - 29, 18 - 28, 18 - 27, 18 - 26, 18 - 25, 18 - 24, 18 - 23, 18 - 22, 18 - 21, 18 - 20, 19 - 30, 19 - 29, 19 - 28, 19 - 27, 19 - 26, 19 - 25, 19 - 24, 19 - 23, 19 - 22, 19 - 21, 19 - 20, 20 - 30, 20 - 29, 20 - 28, 20 - 27, 20 - 26, 20 - 25, 20 - 24, 20 - 23, 20 - 22, 20 - 21, 21 - 30, 21 - 29, 21 - 28, 21 - 27, 21 - 26, 21 - 25, 21 - 24, 21 - 23 or 21 - 22 nucleotides. Ranges and lengths intermediate between the above ranges and lengths are also considered to be part of the present invention.

[0356] In certain preferred embodiments, the length of the complementary region of the antisense sequence to the target sequence and / or the complementary region of the antisense sequence to the sense sequence is at least 17 nucleotides. For example, the length of the complementary region between the antisense strand and the target is 19 to 21 nucleotides, for example, the length of the complementary region is 21 nucleotides.

[0357] In a preferred embodiment, the length of each strand does not exceed 30 nucleotides.

[0358] In certain embodiments, the length of the double-stranded structure of the nucleic acid (e.g., siRNA) is 19 base pairs. In a particularly preferred embodiment, the duplex can have the following structure:

[0359]

[0360] The nucleic acid (e.g., dsRNA) as described herein may also include one or more single-stranded nucleotide overhangs, such as 1 - 4, 2 - 4, 1 - 3, 2 - 3, 1, 2, 3 or 4 nucleotides. The nucleotide overhangs may comprise or consist of nucleoside / nucleoside analogs (including deoxynucleosides / nucleosides). The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. In addition, the nucleotide(s) of the overhang may be present at the 5'-end, 3'-end or both ends of the antisense strand or sense strand of the nucleic acid (e.g., dsRNA).

[0361] In certain preferred embodiments, at least one strand comprises a 3' overhang of at least 1 nucleoside, e.g., at least one strand comprises a 3' overhang of at least 2 nucleosides. The overhang is suitably located on the antisense / guide strand and / or the sense / passenger strand.

[0362] Nucleic Acid Modification

[0363] In certain embodiments, the nucleic acids of the invention, such as RNA (e.g., dsiRNA), do not comprise further modifications, such as chemical modifications or conjugations known in the art and described herein.

[0364] In other preferred embodiments, the nucleic acids of the invention, such as RNA (e.g., dsiRNA), are further chemically modified to enhance stability or other beneficial features.

[0365] In certain embodiments of the invention, substantially all of the nucleosides are modified.

[0366] The nucleic acids referred to in the present invention can be synthesized or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, the content of which is hereby incorporated by reference herein.

[0367] Modifications include, for example, end modifications, such as 5' end modifications (phosphorylation, conjugation, inverted bond) or 3' end modifications (conjugation, DNA nucleosides within RNA or RNA nucleosides within DNA, inverted bond, etc.); base modifications, such as replacement with stabilized bases, destabilized bases or bases that pair with an extended library of pairing partners, conjugated bases; sugar modifications (e.g., at the 2'-position or 4'-position) or sugar replacement; or backbone modifications, including modification or replacement of the phosphodiester bond.

[0368] Specific examples of nucleic acids (e.g., siRNA compounds) useful in the embodiments described herein include, but are not limited to, RNAs containing a modified backbone or lacking native internucleoside linkages. Among them, nucleic acids (e.g., RNA) having a modified backbone include, but are not limited to, those nucleic acids that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified nucleic acids such as RNA that do not have a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides. In some embodiments, the modified nucleic acids such as siRNA have a phosphorus atom in their internucleoside backbone.

[0369] Modified nucleic acids such as RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), thiocarbonylaminophosphates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with opposite polarity (where adjacent nucleoside units are linked 5'-3' or 5'-2'). Also included are various salt, mixed salt, and free acid forms.

[0370] Modified nucleic acids such as RNA can also contain one or more substituted sugar moieties. Nucleic acids such as siRNA (e.g., dsiRNA) mentioned herein can contain at one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted. 2'-O-methyl and 2'-F are preferred modifications.

[0371] In certain preferred embodiments, the nucleic acid contains at least one modified nucleoside.

[0372] The nucleic acids of the invention can contain one or more modified nucleosides on the first strand and / or the second strand.

[0373] In some embodiments, substantially all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand contain modifications.

[0374] In some embodiments, all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand contain modifications.

[0375] In some embodiments, all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand contain modifications.

[0376] In one embodiment, at least one modified nucleoside is selected from the group consisting of: deoxynucleosides, 3'-terminal deoxythymidine (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, conformationally restricted nucleosides, 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'-phosphates, and nucleosides containing 5'-phosphate mimics. In another embodiment, the modified nucleoside comprises a short sequence of 3'-terminal deoxythymidine nucleoside (dT).

[0377] The modification of the nucleoside can preferably be selected from the group consisting of: including but not limited to LNA, HNA, CeNA, 2-methoxyethyl, 2'-O-alkyl, 2-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, and combinations thereof. In another embodiment, the modification of the nucleoside is 2'O-methyl (“2-Me”) or 2'-fluoro modification.

[0378] A preferred modification is the modification of the 2'-OH group of the ribose, optionally selected from 2'-Me or 2'-F modification.

[0379] Preferred nucleic acids contain one or more modified nucleosides in the first strand and / or the second strand to form modified nucleosides, as follows:

[0380] A nucleic acid, wherein the modification is the modification of the 2'-OH group of the ribose, optionally selected from 2'-Me or 2'-F modification.

[0381] A nucleic acid, wherein counting from position 1 of the first strand, the first strand contains a 2'-F modification at any position in position 2, position 6, position 14, or any combination thereof.

[0382] A nucleic acid, wherein counting from position 1 of the second strand, the second strand contains a 2'-F modification at any position in position 7, position 9, position 11, or any combination thereof.

[0383] A nucleic acid, wherein, when counting from position 1 of the second strand, the second strand contains a 2'-F modification at position 7 and / or 9, and / or 11, and / or 13.

[0384] A nucleic acid, wherein, when counting from position 1 of the second strand, the second strand contains 2'-F modifications at positions 7, 9 and 11.

[0385] A nucleic acid, wherein the first strand and the second strand each contain 2'-Me and 2'-F modifications.

[0386] A nucleic acid, which comprises at least one heat-destabilizing modification, when counting from position 1 of the first strand, which is suitably located at one or more positions among positions 1 to 9 of the first strand, and / or suitably located at one or more positions on the second strand aligned with positions 1 to 9 of the first strand, wherein the destabilizing modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid.

[0387] A nucleic acid, wherein, when counting from position 1 of the second strand, the nucleic acid contains 3 or more 2'-F modifications at positions 7 to 13 of the second strand, for example 4, 5, 6 or 7 2'-F modifications at positions 7 to 13 of the second strand.

[0388] A nucleic acid, wherein, when counting from position 1 of the second strand, the second strand contains at least 3, for example 4, 5 or 6 2'-Me modifications at positions 1 to 6 of the second strand.

[0389] A nucleic acid, wherein the first strand contains at least 5 consecutive 2'-Me modifications in the 3'-terminal region (preferably including the terminal nucleoside of the 3'-terminal region), or within at least 1 or 2 nucleosides of the terminal nucleoside of the 3'-terminal region.

[0390] A nucleic acid, wherein the first strand contains 7 consecutive 2'-Me modifications in the 3'-terminal region (preferably including the terminal nucleoside of the 3'-terminal region).

[0391] A nucleic acid, when counting from position 1 of the first strand, which contains at least one heat-destabilizing modification at position 7 of the first strand.

[0392] A nucleic acid, which is an siRNA oligonucleotide, wherein, when counting from position 1 of the second strand, the siRNA oligonucleotide contains at least 3 2'-F modifications at positions 6 to 12 of the second strand.

[0393] A nucleic acid, which is an siRNA oligonucleotide, wherein, when counting from position 1 of the second strand, the second strand contains at least 3 2'-Me modifications at positions 1 to 6 of the second strand.

[0394] A nucleic acid, which is an siRNA oligonucleotide, wherein the first strand and the second strand each comprise an alternating modification pattern, preferably a fully alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleotides of the first strand are modified by: (i) a 2’Me modification on the odd-numbered nucleotides counted from position 1 of the first strand, and (ii) a 2’F modification on the even-numbered nucleotides counted from position 1 of the first strand, and the nucleotides of the second strand are modified by: (i) a 2’F modification on the odd-numbered nucleotides counted from position 1 of the second strand, and (ii) a 2’Me modification on the even-numbered nucleotides counted from position 1 of the second strand. Typically, this fully alternating modification pattern is present in blunt-ended oligonucleotides, wherein each of the first and second strands has a length of 19 or 23 nucleotides.

[0395] Position 1 of the first or second strand is the nucleotide closest to the end of the nucleic acid (ignoring any abasic nucleotides), and this nucleotide is linked to the adjacent nucleotide (at position 2) by a 3’ to 5’ internal bond (referring to the bond between the backbone sugar moieties), and is read in the direction away from the end of the molecule.

[0396] Thus, it can be seen that "position 1 of the sense strand" is the nucleotide closest to the 5’ end at the conventional 5’ end of the sense strand (excluding abasic nucleotides). Typically, the nucleotide at position 1 of the sense strand is equivalent to the 5’ nucleotide of the selected target nucleic acid sequence, and more generally, the sense strand will have nucleotides equivalent to those of the target nucleic acid sequence starting from this position 1 of the sense strand, while also allowing acceptable mismatches between the sequences.

[0397] As used herein, "position 1 of the antisense strand" is the nucleotide closest to the 5’ end at the conventional 5’ end of the antisense strand (excluding abasic nucleotides). As mentioned above, there will be a complementary region between the sense strand and the antisense strand, such that the antisense strand will also have a region complementary to the above-mentioned target nucleic acid sequence.

[0398] In certain embodiments, the nucleic acid such as an RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond. For example, the phosphorothioate or methylphosphonate internucleotide bond can be at the 3'-end or terminal region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands (i.e., the sense strand and the antisense strand).

[0399] In certain embodiments, the phosphorothioate or methylphosphonate internucleotide bond is at the 5'-end or terminal region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands (i.e., the sense strand and the antisense strand).

[0400] In certain embodiments, the phosphorothioate or methylphosphonate internucleotide bond is at the 5'- and 3'-ends or terminal regions of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands (i.e., the sense strand and the antisense strand).

[0401] Any nucleic acid may contain one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS internucleoside bonds at the chain ends.

[0402] At least one oligoribonucleotide chain preferably contains at least two consecutive phosphorothioate modifications among the last three nucleosides of the oligonucleotide.

[0403] Accordingly, the present invention also relates to: a nucleic acid disclosed herein, which contains phosphorothioate internucleoside bonds between at least two or three consecutive positions respectively, such as in the 5' and / or 3' terminal regions and / or near-terminal regions of the second strand, wherein the near-terminal region preferably adjoins the terminal region where one or more abasic nucleosides of the second strand are located.

[0404] A nucleic acid disclosed herein, which contains phosphorothioate internucleoside bonds between at least two or three consecutive positions in the 5' and / or 3' terminal regions of the first strand respectively, wherein preferably, the terminal positions of the 5' and / or 3' terminal regions of the first strand are connected to their adjacent positions by phosphorothioate internucleoside bonds.

[0405] The nucleic acid chain may be RNA containing a phosphorothioate internucleoside bond between three nucleosides, and these three nucleosides are adjacent to two terminal abasic nucleosides.

[0406] A preferred nucleic acid is double-stranded RNA, which contains 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 opposite 3' end of the second strand. Further preferably, the same nucleic acid may also contain a phosphorothioate bond between nucleotides at positions 3-4 and 4-5 of the second strand read from position 1 of the second strand. Further preferably, the same nucleic acid may also contain 2'F modifications at positions 7, 9, 11 of the second strand.

[0407] Preferred modifications of nucleic acids having the following structure are shown below:

[0408]

[0409] A nucleic acid, wherein the modified nucleosides of the first strand have a modification pattern according to the following (5'-3'):

[0410] Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me.

[0411] A nucleic acid, wherein the modified nucleosides of the second strand have a modification pattern according to the following (5'-3'):

[0412] F(s)Me(s)F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F, or

[0413] F-Me-F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F(s)Me(s)F;

[0414] where (s) is a phosphorothioate internucleoside bond.

[0415] A nucleic acid, wherein the modified nucleosides of the second strand have a modification pattern (5'-3') according to the following:

[0416] F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F.

[0417] A nucleic acid, wherein the modified nucleosides of the second strand have a modification pattern (5'-3') according to the following:

[0418] F(s)Me(s)F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F, or

[0419] F-Me-F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F(s)Me(s)F;

[0420] where (s) is a phosphorothioate internucleoside bond.

[0421] A nucleic acid, wherein the modified nucleosides of the second strand have a modification pattern (5'-3') according to the following:

[0422] ia–ia-F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F, or

[0423] F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–ia–ia;

[0424] where ia represents an inverted abasic nucleoside. In certain embodiments, the inverted abasic nucleosides represented by ia-ia are present in two nucleoside overhangs.

[0425] A nucleic acid, wherein the modified nucleosides of the second strand have a modification pattern (5'-3') according to any one of the following:

[0426] ia–ia–F(s)Me(s)F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F, or

[0427] F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me–F–Me(s)F(s)ia–ia;

[0428] where (s) is a phosphorothioate internucleoside bond and ia represents an inverted abasic nucleoside. In certain embodiments, the inverted abasic nucleosides represented by ia-ia are present in two nucleoside overhangs.

[0429] Preferred modifications of nucleic acids having the following structure are shown below:

[0430]

[0431] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0432] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0433] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0434] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0435] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0436] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me.

[0437] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0438] Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0439] Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0440] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0441] Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0442] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0443] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or

[0444] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0445] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0446] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0447] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0448] where (s) is a phosphorothioate internucleoside bond.

[0449] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0450] ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, or

[0451] ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0452] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0453] ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0454] ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, or

[0455] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, or

[0456] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0457] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0458] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, or

[0459] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,

[0460] wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in two nucleoside overhangs.

[0461] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0462] 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

[0463] 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

[0464] 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

[0465] 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

[0466] 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

[0467] 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

[0468] 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

[0469] 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

[0470] 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

[0471] 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,

[0472] Wherein:

[0473] (s) is a phosphorothioate nucleoside internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in a 2-nucleotide overhang.

[0474] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0475] Modification pattern 1: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0476] Or modification pattern 2: Second strand (5'-3'): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0477] Or modification pattern 3: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0478] Or modification pattern 4: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0479] Or modification pattern 5: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0480] Or modification pattern 6: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, 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.

[0481] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0482] Modification pattern 1: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0483] Or modification pattern 2: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0484] Or modification pattern 3: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0485] Or modification pattern 4: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0486] Or modification pattern 5: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0487] Or modification pattern 6: Second strand (5'-3'): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5'-3'): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0488] Where (s) is a phosphorothioate internucleoside linkage.

[0489] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0490] Modification pattern 1: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(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

[0491] Or modification pattern 2: Second strand (5'-3'): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(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

[0492] Or modification pattern 3: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(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

[0493] Or modification pattern 4: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(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

[0494] Or modification pattern 5: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(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

[0495] Or modification pattern 6: Second strand (5'-3'): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(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,

[0496] where (s) is a phosphorothioate internucleoside bond.

[0497] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0498] Modification pattern 1: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0499] Or modification pattern 2: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0500] Or modification pattern 3: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0501] Or modification pattern 4: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0502] Or modification pattern 5: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0503] Or modification pattern 6: Second strand (5’-3’): ia–ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, first strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me,

[0504] wherein ia represents an inverted abasic nucleoside.

[0505] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0506] Modification pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me–Me-ia–ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0507] Or modification pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0508] Or modification pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0509] Or modification pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0510] Or modification pattern 5: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia, First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me

[0511] Or modification pattern 6: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me-ia–ia,- First strand (5'-3'): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me–Me,

[0512] Wherein, ia represents an inverted abasic nucleoside, and when ia–ia represents the presence of an inverted abasic nucleoside at the 3'-end of the second strand, the inverted abasic nucleoside is present in two nucleoside overhangs.

[0513] A nucleic acid, wherein the modified nucleosides include any one of the following modification patterns:

[0514] 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

[0515] 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

[0516] 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

[0517] 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

[0518] 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

[0519] Or modification pattern 6: Second strand (5’-3’): ia–ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me–Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0520] Wherein:

[0521] (s) represents a phosphorothioate internucleoside bond, and ia represents an inverted abasic nucleoside.

[0522] A nucleic acid, wherein the modified nucleosides include any one of the following modification patterns:

[0523] Modification pattern 1: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(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

[0524] Or modification pattern 2: Second strand (5’-3’): Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(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

[0525] Or modification pattern 3: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(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

[0526] Or modification pattern 4: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(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

[0527] Or modification pattern 5: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(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

[0528] Or modification pattern 6: Second strand (5’-3’): Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(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

[0529] Wherein: (s) is a phosphorothioate internucleoside bond, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is present in two nucleoside overhangs.

[0530] A nucleic acid, wherein the first strand comprises a 2’ sugar modification pattern, wherein the modification is at least selected from 2’Me and 2’F sugar modifications, provided that the total number of 2’ sugar modifications in the first strand does not consist of four or six 2’F modifications.

[0531] A nucleic acid, wherein the first strand comprises a 2’ sugar modification pattern, wherein the modification is at least selected from 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.

[0532] A nucleic acid, wherein the first strand comprises a 2’ sugar modification pattern, wherein the modification is at least selected from 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.

[0533] A nucleic acid, wherein the first strand comprises a 2’ sugar modification pattern, wherein the modification is at least selected from 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.

[0534] A nucleic acid, wherein the first strand comprises the following 2’ sugar modification pattern (5’-3’):

[0535] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0536] Wherein X2, X3 and X4 are selected from 2’Me and 2’F sugar modifications, provided that for X2, X3 and X4, at least one is a 2’F sugar modification and the other two sugar modifications are 2’Me sugar modifications.

[0537] A nucleic acid, wherein the first strand comprises the following 2’ sugar modification pattern (5’-3’):

[0538] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0539] Wherein X2 is a 2’F sugar modification, and X3 and X4 are 2’Me sugar modifications.

[0540] A nucleic acid, wherein the first strand comprises the following 2’ sugar modification pattern (5’-3’):

[0541] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0542] Wherein X3 is a 2’F sugar modification, and X2 and X4 are 2’Me sugar modifications.

[0543] A nucleic acid, wherein the first strand comprises the following 2’ sugar modification pattern (5’-3’):

[0544] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0545] Wherein X4 is a 2’F sugar modification, and X2 and X3 are 2’Me sugar modifications.

[0546] A nucleic acid, wherein the first strand comprises a 2’ sugar modification pattern, wherein the modifications are at least selected from 2’Me and 2’F sugar modifications, and the total number of 2’F sugar modifications in the first strand consists of seven 2’F modifications.

[0547] A nucleic acid, wherein the first strand comprises the following 2’ sugar modification pattern (5’-3’):

[0548] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0549] Wherein X2, X3 and X4 are selected from 2’Me and 2’F sugar modifications, provided that for X2, X3 and X4, at least one is a 2’F sugar modification and the other two sugar modifications are 2’Me sugar modifications.

[0550] A nucleic acid, wherein the first strand comprises the following 2’ sugar modification pattern (5’-3’):

[0551] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0552] Wherein X2 is a 2’F sugar modification, and X3 and X4 are 2’Me sugar modifications.

[0553] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0554] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0555] Among them, X3 is 2'F sugar modified, and X2 and X4 are 2'Me sugar modified.

[0556] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0557] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0558] Among them, X4 is 2'F sugar modified, and X2 and X3 are 2'Me sugar modified.

[0559] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0560] Me–F–(Me)3–X1–(Me)7–F–Me–F–(Me)7

[0561] Among them, X1 is a thermal destabilization modification.

[0562] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0563] Me–F–(Me)3–X1–Me–(F)2–(Me)4–F–Me–F–(Me)7

[0564] Among them, X1 is a thermal destabilization modification.

[0565] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0566] (Me)8–(F)3–(Me) 10 .

[0567] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0568] (Me)8–(F)3–(Me) 10 ,and

[0569] wherein the first strand comprises a 2' sugar modification pattern wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, with the proviso that the total number of 2'F sugar modifications in the first strand does not consist of four or six 2'F modifications.

[0570] A nucleic acid, wherein the second strand comprises the following 2'-sugar modification pattern (5'-3'):

[0571] (Me)8–(F)3–(Me) 10 , and

[0572] wherein the first strand comprises a 2'-sugar modification pattern, wherein the modification is selected from at least 2'-Me and 2'-F sugar modifications, and wherein the total number of 2'-F sugar modifications in the first strand consists of three, five or seven 2'-F modifications.

[0573] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0574] (Me)8–(F)3–(Me) 10 , and

[0575] wherein the nucleotides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0576] Me–F–(Me)3–X1–(Me)7–F–Me–F–(Me)7, wherein X1 is a heat-destabilizing modification.

[0577] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0578] (Me)8–(F)3–(Me) 10 , and

[0579] wherein the nucleotides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0580] (Me–F)3–(Me)7–F–Me–F–(Me)7.

[0581] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0582] (Me)8–(F)3–(Me) 10, and

[0583] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0584] Me–F–(Me)3–F–(Me)7–(F–Me)2–F–(Me)5.

[0585] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region having a length of at least 17 nucleosides, and wherein the nucleosides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0586] (Me)8–(F)3–(Me) 10 , and

[0587] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0588] Me–F–(Me)3–F–(Me)7–F–Me–F–(Me)3–F–(Me)3.

[0589] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region having a length of at least 17 nucleosides, and wherein the nucleosides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0590] (Me)8–(F)3–(Me) 10 , and

[0591] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0592] Me–F–(Me)3–X1–Me–(F)2–(Me)4–F–Me–F–(Me)7, wherein X1 is a heat-destabilizing modification.

[0593] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region having a length of at least 17 nucleosides, and wherein the nucleosides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0594] (Me)8–(F)3–(Me) 10 , and

[0595] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0596] (Me–F)3–Me–(F)2–(Me)4–(F–Me)2–(Me)6.

[0597] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0598] (Me)8–(F)3–(Me) 10 , and

[0599] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0600] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–F–(Me)5.

[0601] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2'-sugar modification pattern (5'-3'):

[0602] (Me)8–(F)3–(Me) 10 , and

[0603] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0604] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–(Me)2–F–(Me)3.

[0605] A nucleic acid, wherein the second strand comprises 2'-sugars and has a base-free modification pattern as follows (5'-3'):

[0606] ia-ia-(Me)8–(F)3–(Me) 10 ,

[0607] wherein ia represents an inverted base-free nucleoside.

[0608] A nucleic acid, wherein the second strand comprises 2'-sugars and has a base-free modification pattern as follows (5'-3'):

[0609] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0610] wherein the first strand comprises a 2'-sugar modification pattern, wherein the modification is selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand does not consist of four or six 2'-F modifications.

[0611] A nucleic acid, wherein the second strand comprises a 2'-sugar, and the abasic modification pattern is as follows (5'-3'):

[0612] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0613] wherein the first strand comprises a 2'-sugar modification pattern, wherein 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.

[0614] A nucleic acid, which comprises a first strand that is at least partially complementary to a portion of the 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 duplex region having a length of at least 17 nucleosides, and wherein the nucleosides of the second strand comprise a 2'-sugar, and the abasic modification pattern is as follows (5'-3'):

[0615] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0616] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0617] Me–F–(Me)3–X1–(Me)7–F–Me–F–(Me)7, wherein X1 is a heat-destabilizing modification.

[0618] A nucleic acid, which comprises a first strand that is at least partially complementary to a portion of the 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 duplex region having a length of at least 17 nucleosides, and wherein the nucleosides of the second strand comprise a 2'-sugar, and the abasic modification pattern is as follows (5'-3'):

[0619] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0620] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0621] (Me–F)3–(Me)7–F–Me–F–(Me)7。

[0622] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar, and the abasic modification pattern is as follows (5'-3'):

[0623] ia-ia-(Me)8–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside; and

[0624] wherein the nucleotides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0625] Me–F–(Me)3–F–(Me)7–(F–Me)2–F–(Me)5。

[0626] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar, and the abasic modification pattern is as follows (5'-3'):

[0627] ia-ia-(Me)8–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside; and

[0628] wherein the nucleotides of the first strand comprise the following 2'-sugar modification pattern (5’-3’):

[0629] Me–F–(Me)3–F–(Me)7–F–Me–F–(Me)3–F–(Me)3。

[0630] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the 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 duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar, and the abasic modification pattern is as follows (5’-3’):

[0631] ia-ia-(Me)8–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside; and

[0632] wherein the nucleotides of the first strand comprise the following 2'-sugar modification pattern (5’-3’):

[0633] Me–F–(Me)3–X1–Me–(F)2–(Me)4–F–Me–F–(Me)7,

[0634] wherein X1 is a heat-destabilizing modification.

[0635] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar and have a base-free modification pattern as follows (5'-3'):

[0636] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted base-free nucleoside; and

[0637] wherein the nucleotides of the first strand have a 2'-sugar modification pattern as follows (5'-3'):

[0638] (Me–F)3–Me–(F)2–(Me)4–(F–Me)2–(Me)6.

[0639] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar and have a base-free modification pattern as follows (5'-3'):

[0640] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted base-free nucleoside; and

[0641] wherein the nucleotides of the first strand have a 2'-sugar modification pattern as follows (5'-3'):

[0642] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–F–(Me)5.

[0643] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar and have a base-free modification pattern as follows (5'-3'):

[0644] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted base-free nucleoside; and

[0645] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0646] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–(Me)2–F–(Me)3.

[0647] A nucleic acid, wherein the second strand comprises the following 2'-sugar modification pattern (5'-3'):

[0648] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 ,

[0649] wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond.

[0650] A nucleic acid, wherein the second strand comprises the following 2'-sugar modification pattern (5'-3'):

[0651] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond; and

[0652] wherein the first strand comprises a 2'-sugar modification pattern, wherein the modification is at least selected from 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 four or six 2'-F modifications.

[0653] A nucleic acid, wherein the second strand comprises the following 2'-sugar modification pattern (5'-3'):

[0654] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond; and

[0655] wherein the first strand comprises a 2'-sugar modification pattern, wherein the modification is at least selected from 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.

[0656] A nucleic acid comprising a first strand that is at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a duplex region that is at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise 2'-sugars and have an abasic modification pattern as follows (5'-3'):

[0657] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond, and

[0658] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0659] Me(s)F(s)(Me)3–X1–(Me)7–F–Me–F–(Me)5(s)Me(s)Me, wherein X1 is a heat-destabilizing modification.

[0660] 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 duplex region having a length of at least 17 nucleosides, and wherein the nucleosides of the second strand comprise a 2'-sugar and have an abasic modification pattern as follows (5'-3'):

[0661] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond, and

[0662] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0663] Me(s)F(s)Me–F–Me–F–(Me)7–F–Me–F–(Me)5(s)Me(s)Me.

[0664] 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 duplex region having a length of at least 17 nucleosides, and wherein the nucleosides of the second strand comprise a 2'-sugar and have an abasic modification pattern as follows (5'-3'):

[0665] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond, and

[0666] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0667] Me(s)F(s)(Me)3–F–(Me)7–(F–Me)2–F–(Me)3(s)Me(s)Me.

[0668] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar and have a base modification pattern as follows (5'-3'):

[0669] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond, and

[0670] wherein the nucleotides of the first strand have a 2'-sugar modification pattern as follows (5'-3'):

[0671] Me(s)F(s)(Me)3–F–(Me)7–F–Me–F–(Me)3–F–Me(s)Me(s)Me.

[0672] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar and have a base modification pattern as follows (5'-3'):

[0673] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond, and

[0674] wherein the nucleotides of the first strand have a 2'-sugar modification pattern as follows (5'-3'):

[0675] Me(s)F(s)(Me)3–X1–Me–(F)2–(Me)4–F–Me–F–(Me)5(s)Me(s)Me, where X1 is a heat-destabilizing modification.

[0676] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region that is at least 17 nucleotides in length, and wherein the nucleotides of the second strand comprise a 2'-sugar and have a base modification pattern as follows (5'-3'):

[0677] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond, and

[0678] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0679] Me(s)F(s)Me–F–Me–F–Me–(F)2–(Me)4–(F–Me)2–(Me)4(s)Me(s)Me.

[0680] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise 2'-sugars and have a base-free modification pattern as follows (5'-3'):

[0681] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond, and

[0682] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3'):

[0683] Me(s)F(s)(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–F–(Me)3(s)Me(s)Me.

[0684] A nucleic acid comprising a first strand that is at least partially complementary to a portion of the RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first and second strands form a duplex region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise 2'-sugars and have a base-free modification pattern as follows (5'-3'):

[0685] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 , where ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate bond, and

[0686] wherein the nucleosides of the first strand comprise the following 2'-sugar modification pattern (5'-3):

[0687] Me(s)F(s)(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–(Me)2–F–Me(s)Me(s)Me.

[0688] Preferred modifications are as follows:

[0689] Modification pattern 1:

[0690] 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,

[0691] First strand (5’-3’): Me–F–Me–Me–Me–X1–Me–Me–Me–Me–Me–Me-Me–F–Me–F–Me–Me–Me–Me–Me–Me-Me, where X1 is a heat destabilizing modification;

[0692] Or modification pattern 2:

[0693] 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,

[0694] 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;

[0695] Or modification pattern 3:

[0696] 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,

[0697] 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;

[0698] Or modification pattern 4:

[0699] 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,

[0700] 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;

[0701] Or modification pattern 5:

[0702] 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,

[0703] 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 heat-destabilizing modification;

[0704] Or modification pattern 6:

[0705] 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,

[0706] 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;

[0707] Or modification pattern 7:

[0708] 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,

[0709] 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;

[0710] Or modification pattern 8:

[0711] 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,

[0712] 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.

[0713] Particularly preferred modifications are as follows:

[0714] Modification pattern 1:

[0715] 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,

[0716] 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;

[0717] Or modification pattern 2:

[0718] 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,

[0719] 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;

[0720] Or modification pattern 3:

[0721] 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,

[0722] 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;

[0723] Or modification pattern 4:

[0724] 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,

[0725] 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;

[0726] Or modification pattern 5:

[0727] 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,

[0728] First strand (5’-3’): Me(s)F(s)Me–Me-Me–X1–Me–F–F–Me–Me–Me–Me–F–Me–F–Me–Me–Me–Me–Me(s)Me(s)Me, where X1 is a thermally destabilizing modification;

[0729] Or modification pattern 6:

[0730] 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,

[0731] 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;

[0732] Or modification pattern 7:

[0733] 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,

[0734] 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;

[0735] Or modification pattern 8:

[0736] 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,

[0737] 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;

[0738] where (s) is a phosphorothioate internucleoside linkage.

[0739] Conjugation of Nucleic Acid with Ligand

[0740] Another modification of the nucleic acids of the present invention, such as RNA (e.g., siRNA), involves linking the nucleic acid, such as siRNA, to one or more ligand moieties, for example to enhance the activity, cellular distribution or cellular uptake of the nucleic acid (e.g., siRNA) into cells.

[0741] In some embodiments, the ligand moiety can be linked to the nucleic acid, such as siRNA oligonucleotides, via a cleavable or non-cleavable linker. The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound (e.g., covalently connects two parts of a compound).

[0742] The ligand can be linked to the 3' or 5' end of the sense strand.

[0743] The ligand is preferably conjugated to the 3' end of the sense strand of the nucleic acid (e.g., siRNA agent).

[0744] Accordingly, in another aspect, the present invention relates to a conjugate for inhibiting the expression of a target (e.g., a target gene) in a cell, the conjugate comprising a nucleic acid moiety and one or more ligand moieties, the nucleic acid moiety comprising a nucleic acid as disclosed herein.

[0745] In one aspect, the second strand of the nucleic acid is directly or indirectly (e.g., via a linker) conjugated to one or more ligand moieties, wherein the ligand moiety is generally present in the terminal region of the second strand, preferably in its 3' terminal region.

[0746] In certain embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative linked to the nucleic acid (e.g., dsiRNA) via a linker.

[0747] Accordingly, the present invention relates to a conjugate, wherein the ligand moiety comprises:

[0748] i) one or more GalNAc ligands; and / or

[0749] ii) one or more GalNAc ligand derivatives; and / or

[0750] iii) one or more GalNAc ligands conjugated to the nucleic acid via a linker.

[0751] The GalNAc ligand can be directly or indirectly conjugated to the 5' or 3' terminal region of the second strand of the nucleic acid, preferably conjugated at its 3' terminal region.

[0752] GalNAc ligands are well known in the art and are particularly described in EP3775207A1.

[0753] In some embodiments, the ligand moiety comprises one or more ligands.

[0754] In some embodiments, the ligand moiety comprises one or more saccharide ligands.

[0755] In some embodiments, one or more saccharides can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and / or polysaccharides.

[0756] In some embodiments, one or more saccharides comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0757] In some embodiments, one or more saccharides comprise one or more N-acetyl-galactosamine moieties.

[0758] In some embodiments, the compounds described anywhere herein comprise two or three N-acetylgalactosamine moieties.

[0759] In some embodiments, one or more ligands are linked in a linear configuration or a branched configuration, for example, each configuration is linked to a branch point in the total linker, respectively.

[0760] Exemplary linear configurations and exemplary branched configurations are shown in FIGS. 1a and 1b:

[0761] In FIG. 1a, (linear), (a) and / or (b) can generally represent a linking bond or group, such as a phosphate or phosphorothioate group.

[0762] In FIG. 1b, (branched), in some embodiments, one or more ligands are linked in a biantennary or triantennary branched configuration. Generally, a triantennary branched configuration can be preferred, such as a triantennary N-acetylgalactosamine branched configuration.

[0763] Linker

[0764] Exemplary compounds of the present invention comprise a "linker moiety", such as the "linker moiety" shown in formula (I), which is part of the total "linker".

[0765] Formula I

[0766]

[0767] Wherein: R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl each time it appears;

[0768] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro;

[0769] X1 and X2 are each independently selected from the group consisting of methylene, oxygen and sulfur each time they appear;

[0770] m is an integer from 1 to 6;

[0771] n is an integer from 1 to 10;

[0772] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0773] (i) both q and r cannot be 0 at the same time; and

[0774] (ii) s, t and v cannot all be 0 at the same time;

[0775] Z is an oligonucleoside moiety.

[0776] As further understood in the art, the exemplary compounds of the present invention include the total linker located between the oligonucleoside moiety and the ligand moiety of these compounds. The total linker thus "connects" the oligonucleoside moiety and the ligand moiety to each other.

[0777] The total linker is generally theoretically envisioned as comprising one or more linker building blocks. For example, the linker portion described as the "linker portion" shown in formula (I), which is located near the ligand moiety and generally directly or indirectly connects the ligand moiety to the oligonucleoside moiety through a branching point. The linker portion described in formula (I) is also often referred to as the "ligand arm" (one or more) of the total linker. There may also (but not always) be another linker portion between the oligonucleoside moiety and the branching point, which is generally referred to as the "tether moiety" of the total linker, which "tethers" the oligonucleoside moiety to the rest of the conjugate compound. Such "ligand arms" and / or "linker portions" and / or "tether moieties" can be envisioned with reference to the linear and / or branched configurations described above.

[0778] As can be seen from the claims and the rest of the patent specification, the scope of the present invention extends to linear or branched configurations and there is no limitation on the number of individual ligands that may be present. In addition, those skilled in the art will also realize that according to the prior art and the expertise of oligonucleoside chemists, there are many structures that can be used as linker portions.

[0779] The remainder of the linker (except for the joint portion) listed in the claims, as well as the remainder of the patent specification, are shown by their chemical composition in formula (I), which the inventors consider to be particularly unique to the present invention. However, in more general terms, these chemical compositions can be described as the "tether portion" as previously described, where the "tether portion" is part of the linker and contains the atomic group between Z (i.e., the oligonucleotide portion) and the joint portion as described in formula (I).

[0780] The tether portion of formula I

[0781] Regarding formula (I), the "tether portion" contains the atomic group between Z (i.e., the oligonucleotide portion) and the joint portion.

[0782] In some embodiments, R1 is hydrogen each time it appears. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl.

[0783] In some embodiments, R2 is hydroxyl. In some embodiments, R2 is halogen. In some embodiments, R2 is fluorine. In some embodiments, R2 is chlorine. In some embodiments, R2 is bromine. In some embodiments, R2 is iodine. In some embodiments, R2 is nitro.

[0784] In some embodiments, X1 is methylene. In some embodiments, X1 is oxygen. In some embodiments, X1 is sulfur.

[0785] In some embodiments, X2 is methylene. In some embodiments, X2 is oxygen. In some embodiments, X2 is sulfur.

[0786] In some embodiments, m = 3.

[0787] In some embodiments, n = 6.

[0788] In some embodiments, X1 is oxygen and X2 is methylene. In some embodiments, both X1 and X2 are methylene.

[0789] 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.

[0790] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is fluorine, X2 is methylene, v = 1, t = 1, s = 1, X1 is methylene, q = 1 and r = 2.

[0791] Thus, in some embodiments, the exemplary compounds of the present invention comprise the following structure:

[0792]

[0793] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is fluorine, X2 is methylene, v = 1, t = 1, s = 1, X1 is oxygen, q = 1 and r = 2.

[0794] Thus, in some embodiments, the exemplary compounds of the present invention comprise the following structure:

[0795]

[0796] Alternative tether portion

[0797] During the synthesis of the compounds of the present invention, alternative tether portion structures may occur. In some embodiments, the alternative tether portion has a change in one or more atoms in the tether portion of the total linker compared to the tether portions described anywhere herein.

[0798] In some embodiments, the alternative tether portion is a compound of formula (I) described anywhere herein, wherein R2 is hydroxy.

[0799] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is hydroxy, X2 is methylene, v = 1, t = 1, s = 1, X1 is methylene, q = 1 and r = 2.

[0800] Thus, in some embodiments, the compounds of the present invention comprise the following structure:

[0801]

[0802] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is hydroxy, X2 is methylene, v = 1, t = 1, s = 1, X1 is oxygen, q = 1 and r = 2.

[0803] Thus, in some embodiments, the compounds of the present invention comprise the following structure:

[0804]

[0805] Linker portion

[0806] With respect to formula (I), the "linker portion" described in formula (I) comprises a group of atoms located between the tether portion described anywhere herein and the ligand portion described anywhere herein.

[0807] In some embodiments:

[0808] The portion described in formula (I) as described anywhere herein:

[0809]

[0810] is any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa):

[0811]

[0812] Wherein:

[0813] A I is hydrogen, or a suitable hydroxyl protecting group;

[0814] a is an integer of 2 or 3; and

[0815] b is an integer from 2 to 5; or

[0816]

[0817] Wherein:

[0818] A I is hydrogen, or a suitable hydroxyl protecting group;

[0819] a is an integer of 2 or 3; and

[0820] c and d are independently integers from 1 to 6; or

[0821]

[0822] Wherein:

[0823] A I is hydrogen, or a suitable hydroxyl protecting group;

[0824] A is 2 or 3; and

[0825] e is an integer from 2 to 10.

[0826] In some embodiments, the moiety described in formula (I):

[0827]

[0828] is formula (VIa):

[0829]

[0830] Wherein:

[0831] A I is hydrogen, or a suitable hydroxyl protecting group;

[0832] A is 3; and

[0833] B is the integer 3.

[0834] In some embodiments, the moiety described in formula (I) as described anywhere herein:

[0835]

[0836] is of formula (VII):

[0837]

[0838] wherein:

[0839] A I is hydrogen;

[0840] A is an integer from 2 or 3, preferably 3.

[0841] Other exemplary compounds of the present invention include a "linker moiety" as shown in formula (I*), which is part of the total "linker".

[0842]

[0843] wherein:

[0844] r and s are independently integers selected from 1 to 16; and

[0845] Z is an oligonucleoside moiety.

[0846] As further understood in the art, the exemplary compounds of the present invention contain a total linker located between the oligonucleoside moiety and the ligand moiety of these compounds. The total linker thus "connects" the oligonucleoside moiety and the ligand moiety to each other.

[0847] The total linker is generally theoretically envisioned as containing one or more linker building blocks. For example, for example, a linker moiety described as a "linker moiety" as shown in formula (I*), which is located near the ligand moiety and generally directly or indirectly connects the ligand moiety to the oligonucleoside moiety through a branching point. The linker moiety described in formula (I*) is also often referred to as the "ligand arm" (s) of the total linker. There may also (but not always) be another linker moiety between the oligonucleoside moiety and the branching point, which is generally referred to as the "tethering moiety" of the total linker, which "tethers" the oligonucleoside moiety to the rest of the conjugate compound. Such "ligand arms" and / or "linker moieties" and / or "tethering moieties" can be envisioned with reference to the linear and / or branched configurations described above.

[0848] As can be seen from the remainder of the claims and the patent specification, the scope of the present invention extends to linear or branched configurations and there is no limitation on the number of individual ligands that may be present. In addition, those skilled in the art will also recognize that, based on the prior art and the expertise of oligonucleoside chemists, there are many structures that can be used as linker moieties.

[0849] The remainder of the linker (except for the linker moiety) listed in the claims, as well as the remainder of the patent specification, are shown by their chemical composition in formula (I), which the inventors believe is particularly unique to the present invention. However, in more general terms, these chemical compositions can be described as the "tethering moiety" as previously described, where the "tethering moiety" is part of the linker and contains the atomic group between Z (i.e., the oligonucleotide moiety) and the linker moiety as described in formula (I).

[0850] Tethering moiety

[0851] Regarding formula (I*), the "tethering moiety" contains the atomic group between Z (i.e., the oligonucleotide moiety) and the linker moiety.

[0852] In some embodiments, s is an integer selected from 4 to 12. In some embodiments, s is 6.

[0853] In some embodiments, r is an integer selected from 4 to 14. In some embodiments, r is 6. In some embodiments, r is 12.

[0854] In some embodiments, r is 12 and s is 6.

[0855] Thus, in some embodiments, the exemplary compounds of the present invention comprise the following structure:

[0856]

[0857] In some embodiments, r is 6 and s is 6.

[0858] Thus, in some embodiments, the exemplary compounds of the present invention comprise the following structure:

[0859]

[0860] Linker moiety

[0861] Regarding formula (I*), the "linker moiety" described in formula (I) contains the atomic group located between the tethering moiety described anywhere herein and the ligand moiety described anywhere herein.

[0862] In some embodiments, the moiety described in formula (I*) as described anywhere herein:

[0863]

[0864] Is any one of formula (IV*), (V*), or (VI*), preferably formula (IV*):

[0865]

[0866] Wherein:

[0867] A I is hydrogen, or a suitable hydroxyl protecting group;

[0868] a is an integer of 2 or 3; and

[0869] b is an integer from 2 to 5; or

[0870]

[0871] Wherein:

[0872] A I is hydrogen, or a suitable hydroxyl protecting group;

[0873] a is an integer of 2 or 3; and

[0874] c and d are independently integers from 1 to 6; or

[0875]

[0876]

[0877] Wherein:

[0878] A I is hydrogen, or a suitable hydroxyl protecting group;

[0879] a is an integer of 2 or 3; and

[0880] e is an integer from 2 to 10.

[0881] In some embodiments, the moiety described in formula (I):

[0882]

[0883] is of formula (VIa*):

[0884]

[0885] Wherein:

[0886] A I is hydrogen, or a suitable hydroxyl protecting group;

[0887] a is 3; and

[0888] b is the integer 3.

[0889] In some embodiments, the moiety described in formula (I) as described anywhere herein:

[0890]

[0891] is of formula (VII*):

[0892]

[0893] wherein:

[0894] A I is hydrogen;

[0895] a is an integer of 2 or 3.

[0896] In some embodiments, a = 2. In some embodiments, a = 3. In some embodiments, b = 3.

[0897] Vectors and Cells

[0898] In one aspect, the present invention provides a cell containing a nucleic acid, such as the inhibitory RNA [RNAi] described herein.

[0899] In one aspect, the present invention provides a cell comprising the vector described herein.

[0900] In one aspect, the present invention provides a vector comprising an oligonucleotide inhibitor such as iRNA (e.g., siRNA).

[0901] Pharmaceutically Acceptable Composition

[0902] In one aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of a target gene, the composition comprising an inhibitor disclosed herein such as an oligomer, e.g., a nucleic acid.

[0903] The pharmaceutically acceptable composition may comprise an excipient and / or a carrier.

[0904] Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) fillers such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (22) other non-toxic and compatible substances for pharmaceutical formulations.

[0905] Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethyl cellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0906] Pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with the nucleic acid and are suitable for parenteral administration can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxyethyl cellulose, polyvinylpyrrolidone, etc.

[0907] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents (e.g., alcohols), or solutions of nucleic acids in liquid or solid oil bases. The solutions can also contain buffering agents, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with the nucleic acid and are suitable for parenteral administration can be used.

[0908] 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 buffered solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffered solution can be phosphate buffered saline (PBS).

[0909] Dose

[0910] The pharmaceutical compositions of the invention can be administered in a dose sufficient to inhibit gene expression or alter the expression or function of a target (e.g., lncRNA). Generally, when the composition comprises a nucleic acid, a suitable dose of the nucleic acid (e.g., siRNA) of the invention will range from about 0.001 to about 200.0 milligrams per kilogram of body weight per day of the recipient, typically in the range of about 1 to 50 mg per kilogram of body weight per day. Generally, a suitable dose of the nucleic acid (e.g., siRNA) of the invention will range from about 0.1 mg / kg to about 5.0 mg / kg, for example, about 0.3 mg / kg and about 3.0 mg / kg.

[0911] A repeated dose regimen can include periodic (e.g., every other day or once a year) administration of a therapeutically effective amount of a nucleic acid, such as siRNA. In certain embodiments, the nucleic acid (e.g., siRNA) is administered about once a month to about once a quarter (i.e., about once every three months).

[0912] In various embodiments, the nucleic acid (e.g., siRNA agent) is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or 0.5 mg / kg to about 50 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA agent) is administered at a dose of about 10 mg / kg to about 30 mg / kg. In certain embodiments, the nucleic acid (e.g., siRNA agent) is administered at a dose selected from: about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg. In certain embodiments, the nucleic acid (e.g., siRNA agent) is administered about once a week, once a month, once every two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg / kg to about 5.0 mg / kg. In certain embodiments, the nucleic acid (e.g., siRNA agent) is administered to a subject once a week. In certain embodiments, the nucleic acid (e.g., siRNA agent) is administered to a subject once a month. In certain embodiments, the nucleic acid (e.g., siRNA agent) is administered once a quarter (i.e., once every three months).

[0913] After an initial treatment regimen, treatment can be carried out at a lower frequency. For example, after administering once a week or once every two weeks for three months, it can be repeated monthly for six months or one year; or for even longer.

[0914] The pharmaceutical composition can be administered once daily, or in two, three or more sub-doses at appropriate intervals during the day, or even by continuous infusion or delivery via a controlled-release formulation. In this case, the nucleic acid (e.g., siRNA) contained in each sub-dose must be correspondingly smaller to achieve the total daily dose. The dosage units can also be combined, for example, using a conventional sustained-release formulation to deliver over several days, which can provide a sustained release of the nucleic acid (e.g., siRNA) over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering agents at a specific site, for example, they can be used together with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0915] In other embodiments, a single dose of the pharmaceutical composition can be long-acting such that subsequent doses are administered at intervals of no more than 3, 4 or 5 days, or at intervals of no more than 1, 2, 3 or 4 weeks. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In other embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once every two months. In certain embodiments, siRNA is administered at about once a month to about once a quarter (i.e., about once every three months), or even once every 6 months or 12 months.

[0916] As is known in the art, conventional methodologies or in vivo tests based on the use of suitable animal models can be used to evaluate the effective dose and in vivo half-life of individual nucleic acids (e.g., siRNA) covered by the present invention.

[0917] Depending on whether local or systemic treatment is required and the area to be treated, the pharmaceutical composition of the present invention can be administered in a variety of ways. Administration can be local (e.g., via a transdermal patch), pulmonary administration, e.g., by inhalation or insufflation of a powder or aerosol, including via a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous administration, e.g., via an implant device; or intracranial administration, e.g., via intracerebral, 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.

[0918] In one embodiment, the nucleic acid (e.g., siRNA agent) is administered subcutaneously to a subject.

[0919] Inhibitors such as nucleic acids (e.g., siRNA) can be delivered in a manner that targets specific tissues (e.g., particularly hepatocytes).

[0920] Method for Inhibiting Gene Expression or Inhibiting Target Expression or Function

[0921] The present invention also provides methods for inhibiting gene expression in cells and methods for inhibiting the expression and / or function of other target molecules (e.g., lncRNA). These methods include contacting the cells with an amount of a nucleic acid (e.g., an siRNA agent, e.g., a double-stranded siRNA reagent) of the present invention that effectively inhibits the expression of a gene in the cells, thereby inhibiting the expression of the gene in the cells. In a preferred embodiment, the gene encodes an enzyme involved in post-translational glycosylation. In a more preferred embodiment, the gene is B4GALT1.

[0922] Contacting the cells with the inhibitor (e.g., a nucleic acid such as siRNA, e.g., a double-stranded siRNA agent) can be carried out in vitro or in vivo. Contacting the cells with the inhibitor nucleic acid (e.g., siRNA) in vivo includes contacting cells or cell populations in a subject (e.g., a human subject) with the nucleic acid (e.g., siRNA). Combinations of methods for contacting cells in vitro and in vivo are also possible. As described above, the cells can be contacted directly or indirectly. In addition, contacting the cells can be achieved through a targeting ligand moiety (including any ligand moiety described herein or known in the art). In a preferred embodiment, the targeting ligand moiety is a carbohydrate moiety, e.g., a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to the site of interest.

[0923] As used herein, the terms "inhibiting\suppressing" can be used interchangeably with "reducing", "silencing", "downregulating", and other similar terms, and include any level of inhibition.

[0924] In some embodiments of the methods of the present invention, the expression or activity of the gene or the inhibitory target (e.g., lncRNA) is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or is inhibited to below the measured detection level, preferably when determined by qPCR as described herein and / or when siRNA is introduced into target cells by transfection. In certain embodiments, the method includes clinically relevant inhibition of target gene expression, e.g., as demonstrated by clinically relevant outcomes after treating a subject with an agent to reduce gene expression and / or target activity.

[0925] In some embodiments, when transfected into cells, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an IC50 value of less than 2500 pM, 2400 pM, 2300 pM, 2200 pM, 2100 pM, 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM, as described herein, preferably determined by qPCR and more preferably by reverse transcriptase (RT)-qPCR.

[0926] In a preferred embodiment, when transfected into cells, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an IC50 value of less than 2500 pM. In a more preferred embodiment, when transfected into cells, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an IC50 value of less than 1000 pM. In an even more preferred embodiment, when transfected into cells, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an IC50 value of less than 500 pM. In a most preferred embodiment, when transfected into cells, the nucleic acids of the invention inhibit the expression of the B4GALT1 gene with an IC50 value of less than 100 pM.

[0927] The inhibition of B4GALT1 gene expression can be quantified using the following methods:

[0928] Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB cell bank) can be cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS under an atmosphere of 37 °C and 5% CO2. Then, the cells were transfected with 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)), using 10x3-fold serial dilutions with a final duplex concentration ranging from 20 nM to 1 pM. Transfection can be carried out by adding 9.7 μL of Opti-MEM (ThermoFisher) and 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture was incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells were incubated at 37 °C / 5% CO2 for 24 hours, and then total RNA was purified using the RNeasy 96 kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in a single experiment.

[0929] cDNA synthesis can be carried out using the FastQuant RT (with gDNA enzyme) kit (Tiangen). Real-time quantitative PCR (qPCR) can be carried out using the TaqMan Gene Expression Assay kit (ThermoFisher Scientific) with specific primers for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1) on an ABIPrism 7900HT or an ABI QuantStudio 7.

[0930] qPCR can be performed in duplicate on cDNA from each well, and the average cycle threshold (Ct) can be calculated. The relative B4GALT1 expression can be calculated according to the average Ct value using the comparative Ct (ΔΔCt) method, normalized to GAPDH and relative to untreated cells. The maximum percentage inhibition and IC50 value of B4GALT1 expression can be calculated using the four-parameter (variable slope) model with GraphPad Prism 9.

[0931] Alternatively or additionally, the inhibitory potential of the nucleic acids of the present invention can be quantified without pre-transfecting the target cells with the said nucleic acids.

[0932] Thus, in some 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 of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, or 100 nM, as determined herein, preferably by qPCR, and more preferably by reverse transcriptase (RT)-qPCR.

[0933] 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 of less 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 of less 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 of less than 200 nM. In 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 of less than 100 nM.

[0934] The following methods can be used to quantify the inhibition of B4GALT1 gene expression in the presence of free nucleic acids:

[0935] Primary C57BL / 6 mouse hepatocytes (PMH) can be freshly isolated by a two-step collagenase liver perfusion method. The cells can be cultured in DMEM (Gibco - 11995 - 092) supplemented with FBS, penicillin / streptomycin, HEPES, and L-glutamine. The cells can be cultured in a humidified incubator at 37 °C in an atmosphere of 5% CO2. Within 2 hours after isolation, PMH can be seeded at a density of 36,000 cells / well in a conventional 96-well tissue culture plate. The dose-response analysis in PMH can be performed by directly incubating the cells in a gymnotic free uptake environment, with the final concentration of GalNAc-siRNA being 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM. In the control wells, the cells can be incubated without GalNAc-siRNA. After 48 hours of incubation, the cells are harvested for RNA extraction. Total RNA can be extracted using an RNeasy kit according to the manufacturer's instructions (Qiagen, Shanghai, China). After reverse transcription, real-time quantitative PCR is performed using an 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. This method measures the Ct difference (ΔCt) between the target gene and the housekeeping gene. 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 。

[0936] Alternatively or additionally, the inhibition of B4GALT1 gene expression can be characterized by a decrease in the average relative expression of the B4GALT1 gene.

[0937] In some embodiments, when the cells are transfected with 0.1 nM of the nucleic acid of the present invention, the average relative expression of B4GALT1 is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4, as described herein, preferably determined by qPCR, more preferably determined by reverse transcriptase (RT)-qPCR.

[0938] In some embodiments, when the cells are transfected with 5 nM of the nucleic acid of the present invention, the average relative expression of B4GALT1 is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3, as described herein, preferably determined by qPCR, more preferably determined by reverse transcriptase (RT)-qPCR.

[0939] The average relative expression of the B4GALT1 gene can be quantified using the following method:

[0940] Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB cell bank) can be cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS under an atmosphere of 37 °C and 5% CO2. Cells were transfected with 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)), and the final duplex concentration was 5 nM and 0.1 nM. Transfection can be carried out by adding 9.7 μL of Opti-MEM (ThermoFisher) and 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. Incubate the mixture at room temperature for 15 minutes, then add 100 μL of complete growth medium containing 20,000 Huh7 cells. Incubate the cells at 37 °C / 5% CO2 for 24 hours, and then purify the total RNA using the RNeasy 96 kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in two separate experiments.

[0941] cDNA synthesis can be performed using the FastQuant RT (with gDNA enzyme) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed using the TaqMan Gene Expression Assay kit (ThermoFisher Scientific) with specific primers for human B4GALT1 (Hs00155245_m1) and human GAPDH (Hs02786624_g1) on an ABIPrism 7900HT or ABI QuantStudio 7.

[0942] qPCR can be performed in duplicate on cDNA from each well, and the average Ct can be calculated. The relative B4GALT1 expression can be calculated using the comparative Ct (ΔΔCt) method based on the average Ct value, normalized to GAPDH and relative to untreated cells.

[0943] Inhibition of gene expression can be manifested by a decrease in the amount of mRNA of the target gene compared to a suitable control. Inhibition of target function can be manifested by a decrease in target activity compared to a suitable control.

[0944] In other embodiments, inhibition of gene or other target expression can be evaluated by a decrease in parameters related to gene expression function (such as protein expression or signal transduction pathways).

[0945] Method for Treating or Preventing Diseases Associated with Gene Expression / Target Such as LCNRNA Functional Expression

[0946] The present invention also provides a method for reducing or inhibiting gene expression in a cell or reducing the expression or function of a target using the nucleic acid of the present invention (e.g., siRNA) or a composition comprising the nucleic acid of the present invention (e.g., siRNA). The method comprises contacting the cell with the nucleic acid of the present invention (e.g., siRNA) and maintaining the cell for a sufficient time to obtain degradation of the mRNA transcript of the gene, thereby inhibiting gene expression in the cell. Reduction of the gene expression or function of the target 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.

[0947] In the method of the present invention, the cell can be contacted in vitro or in vivo, i.e., the cell can be in a subject.

[0948] Cells suitable for treatment using the method of the present invention can be any cell that expresses a gene of interest or a target of interest associated with a disease.

[0949] The in vivo method of the present invention can comprise administering to a subject a composition comprising the nucleic acid of the present invention (e.g., siRNA), wherein the nucleic acid (e.g., siRNA) comprises a nucleoside sequence complementary to at least a portion of the RNA transcript of a mammalian gene to be treated, or a nucleoside sequence complementary to another nucleic acid whose expression and / or function is associated with a disease.

[0950] The present invention also provides a method for treating a subject in need thereof. The treatment method of the present invention comprises administering to a subject (e.g., a subject who can benefit from reducing or inhibiting gene expression and / or the expression and / or function of a target) a nucleic acid of the present invention (e.g., siRNA) in a therapeutically effective amount, such as a target gene nucleic acid (e.g., siRNA) or a pharmaceutical composition comprising a nucleic acid comprising a target gene.

[0951] The nucleic acid of the present invention (e.g., siRNA) can be administered as a "naked" nucleic acid or "naked" siRNA, and can be administered without a pharmaceutical composition. The naked nucleic acid can be in a suitable buffer solution. The buffer solution can comprise acetate, citrate, gliadin, 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 such that it is suitable for administration to a subject.

[0952] Alternatively, the nucleic acid of the present invention (e.g., siRNA) can be administered as a pharmaceutical composition (e.g., a dsiRNA liposomal formulation).

[0953] In one embodiment, the method comprises administering the composition described herein such that the expression of the target gene is reduced for, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the expression of the target gene is reduced for an extended duration, e.g., at least about two days, three days, four days, or more days, e.g., about one week, two weeks, three weeks, or four weeks or longer, e.g., about 1 month, 2 months, or 3 months.

[0954] A therapeutically effective amount of a nucleic acid (e.g., siRNA) can be administered to a subject, e.g., from about 0.01 mg / kg to about 200 mg / kg.

[0955] The nucleic acid (e.g., siRNA) can be administered by intravenous infusion periodically over a period of time. In certain embodiments, after an initial treatment regimen, treatment can be performed at a lower frequency. Administration of the siRNA can reduce the level of the gene product in the target gene, e.g., reduce the level of the gene product in the target gene in a patient's cells or tissues by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay used. In certain embodiments, administration causes clinical stabilization or preferably a clinically relevant reduction of at least one sign or symptom of the gene-related disorder.

[0956] Alternatively, the nucleic acid (e.g., siRNA) can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver the desired daily dose of the nucleic acid (e.g., siRNA) to the subject. The injection can be repeated over a period of time. The administration can be repeated periodically. In certain embodiments, after an initial treatment regimen, treatment can be performed at a lower frequency. The repeated dose regimen can include periodic administration of a therapeutically effective amount of the nucleic acid, e.g., every other day or once a year. In certain embodiments, the nucleic acid is administered from about once a month to about once a quarter (i.e., about once every three months).

[0957] In one aspect, the present invention can be applied to the compounds, methods, compositions, or uses numbered 1-101 below (wherein any reference to any formula in items 1-101 refers only to those formulas defined within items 1-101, which are reproduced in Figure 6 ).

[0958] 1. A compound comprising the following structure:

[0959]

[0960] Wherein:

[0961] R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl each time it appears;

[0962] R2 is selected from the group consisting of: hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro;

[0963] X1 and X2 are each independently selected from the group consisting of: methylene, oxygen and sulfur;

[0964] m is an integer from 1 to 6;

[0965] n is an integer from 1 to 10;

[0966] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0967] (i) both q and r cannot be 0 at the same time; and

[0968] (ii) s, t and v cannot all be 0 at the same time;

[0969] Z is an oligonucleotide moiety.

[0970] 2. The compound according to item 1, wherein R1 is hydrogen each time it appears.

[0971] 3. The compound according to item 1, wherein R1 is methyl.

[0972] 4. The compound according to item 1, wherein R1 is ethyl.

[0973] 5. The compound according to any one of items 1 to 4, wherein R2 is hydroxy.

[0974] 6. The compound according to any one of items 1 to 4, wherein R2 is halogen.

[0975] 7. The compound according to item 6, wherein R2 is fluorine.

[0976] 8. The compound according to item 6, wherein R2 is chlorine.

[0977] 9. The compound according to item 6, wherein R2 is bromine.

[0978] 10. The compound according to item 6, wherein R2 is iodine.

[0979] 11. The compound according to item 6, wherein R2 is nitro.

[0980] 12. The compound according to any one of items 1 to 11, wherein X1 is methylene.

[0981] 13. The compound according to any one of items 1 to 11, wherein X1 is oxygen.

[0982] 14. A compound according to any one of items 1 to 11, wherein X1 is sulfur.

[0983] 15. A compound according to any one of items 1 to 14, wherein X2 is methylene.

[0984] 16. A compound according to any one of items 1 to 15, wherein X2 is oxygen.

[0985] 17. A compound according to any one of items 1 to 16, wherein X2 is sulfur.

[0986] 18. A compound according to any one of items 1 to 17, wherein m = 3.

[0987] 19. A compound according to any one of items 1 to 18, wherein n = 6.

[0988] 20. A compound according to items 13 and 15, wherein X1 is oxygen and X2 is methylene, and preferably wherein:

[0989] q = 1,

[0990] r = 2,

[0991] s = 1,

[0992] t = 1,

[0993] v = 1.

[0994] 21. A compound according to items 12 and 15, wherein both X1 and X2 are methylene, and preferably wherein:

[0995] q = 1,

[0996] r = 3,

[0997] s = 1,

[0998] t = 1,

[0999] v = 1.

[1000] 22. A compound according to any one of items 1 to 21, wherein Z is:

[1001]

[1002] wherein:

[1003] Z1, Z2, Z3, Z4 are each independently oxygen or sulfur upon each occurrence; and one of the bonds between P and Z2 and between P and Z3 is a single bond and the other is a double bond.

[1004] 23. A compound according to item 22, wherein the oligonucleotide is an RNA compound capable of regulating, preferably inhibiting, the expression of a target gene.

[1005] 24. A compound according to item 23, wherein the RNA compound comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end.

[1006] 25. A compound according to item 24, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[1007] 26. A compound according to item 24, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[1008] 27. A compound of formula (II):

[1009]

[1010] 28. A compound of formula (III):

[1011]

[1012] 29. A compound according to item 27 or 28, wherein the oligonucleotide comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1013] 30. A composition comprising a compound of formula (II) as defined in item 27, and a compound of formula (III) as defined in item 28, optionally depending on item 29.

[1014] 31. A composition according to item 30, wherein the compound of formula (III) as defined in item 28 is present in an amount in the range of 10 to 15% by weight of the composition.

[1015] 32. A compound of formula (IV):

[1016]

[1017] 33. A compound of formula (V):

[1018]

[1019] 34. A compound according to item 32 or 33, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1020] 35. A composition comprising a compound of formula (IV) as defined in item 32, and a compound of formula (V) as defined in item 33, optionally depending on item 34.

[1021] 36. A composition according to item 35, wherein the compound of formula (V) as defined in item 33 is present in the composition in an amount of 10 to 15% by weight.

[1022] 37. A compound according to any one of items 1 to 29 or 32 to 34, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex further comprising one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[1023] 38. A compound according to item 37, wherein the modification is selected from: 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[1024] 39. A compound according to any one of items 1 to 29, or 32 to 34, or 37 to 38, wherein the oligonucleoside comprises one or more degradation protection moieties at one or more ends.

[1025] 40. A compound according to item 39, wherein the one or more degradation protection moieties are not present at the ends of the oligonucleoside chain carrying the ligand moiety, and / or wherein the one or more degradation protection moieties are selected from: phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein the inverted abasic nucleoside is present at the distal end of the chain carrying the ligand moiety.

[1026] 41. A compound according to any one of items 1 to 29, or 32 to 34, or 37 to 40, wherein the ligand moiety described in formula (I) in item 1 comprises one or more ligands.

[1027] 42. A compound according to item 41, wherein the ligand moiety described in formula (I) in item 1 comprises one or more carbohydrate ligands.

[1028] 43. A compound according to item 42, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[1029] 44. A compound according to item 43, wherein said one or more saccharides comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[1030] 45. A compound according to item 44, wherein said one or more saccharides comprise one or more N-acetyl-galactosamine moieties.

[1031] 46. A compound according to item 45, which comprises two or three N-acetylgalactosamine moieties.

[1032] 47. A compound according to any one of items 41 to 46, wherein said one or more ligands are linked in a linear configuration or a branched configuration.

[1033] 48. A compound according to item 47, wherein said one or more ligands are linked in a biantennary or triantennary branched configuration.

[1034] 49. A compound according to items 46 to 48, wherein the moiety described in formula (I) in item 1:

[1035]

[1036] is any one of formula (VIa), (VIb), or (VIc), preferably formula (VIa):

[1037]

[1038] wherein:

[1039] A I is hydrogen, or a suitable hydroxyl protecting group;

[1040] a is an integer of 2 or 3; and

[1041] b is an integer from 2 to 5; or

[1042]

[1043] wherein:

[1044] A I is hydrogen, or a suitable hydroxyl protecting group;

[1045] a is an integer of 2 or 3; and

[1046] c and d are independently integers from 1 to 6; or

[1047]

[1048] wherein:

[1049] A Iis hydrogen, or a suitable hydroxyl protecting group;

[1050] a is an integer of 2 or 3; and

[1051] e is an integer from 2 to 10.

[1052] 50. A compound according to items 46 to 48, wherein the moiety described by formula (I) in item 1:

[1053]

[1054] is formula (VII):

[1055]

[1056] wherein:

[1057] A I is hydrogen;

[1058] a is an integer of 2 or 3.

[1059] 51. A compound according to item 49 or 50, wherein a = 2.

[1060] 52. A compound according to item 49 or 50, wherein a = 3.

[1061] 53. A compound according to item 49, wherein b = 3.

[1062] 54. A compound of formula (VIII):

[1063]

[1064] 55. A compound of formula (IX):

[1065]

[1066] 56. A compound according to item 54 or 55, wherein the oligonucleotide comprises an RNA duplex, the RNA duplex comprises a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1067] 57. A composition comprising a compound of formula (VIII) as defined in item 54, and a compound of formula (IX) as defined in item 55, optionally depending on item 56.

[1068] 58. A composition according to item 57, wherein the compound of formula (IX) as defined in item 55 is present in the composition in an amount of 10 to 15% by weight.

[1069] 59. Compound of formula (X):

[1070]

[1071] 60. Compound of formula (XI):

[1072]

[1073] 61. The compound according to item 59 or 60, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprises a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5' and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1074] 62. A composition comprising a compound of formula (X) as defined in item 59, and a compound of formula (XI) as defined in item 60, optionally depending on item 61.

[1075] 63. The composition according to item 62, wherein the compound of formula (XI) as defined in item 60 is present in an amount of 10 to 15% by weight of the composition.

[1076] 64. The compound as defined in any one of items 54 to 63, wherein the oligonucleoside comprises an RNA duplex, and the RNA duplex further comprises one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[1077] 65. The compound according to item 64, wherein the modification is selected from: 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[1078] 66. The compound according to any one of items 54 to 65, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more ends.

[1079] 67. The compound according to item 66, wherein the one or more degradation protection moieties are not present at the ends of the oligonucleoside chain carrying the ligand moiety, and / or wherein the one or more degradation protection moieties are selected from: phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein as shown in any one of formula (VIII), (IX), (X), or (XI) in any one of items 54, 55, 59, or 60, the inverted abasic nucleoside is present at the distal end of the chain carrying the ligand moiety.

[1080] 68. A method for preparing a compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67 and / or a composition according to any one of items 30, 31, 35, 36, 57, 58, 62, 63, which comprises reacting compounds of formula (XII) and (XIII):

[1081]

[1082] wherein:

[1083] Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1084] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro;

[1085] Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen and sulfur;

[1086] M is an integer from 1 to 6;

[1087] n is an integer from 1 to 10;

[1088] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1089] (i) q and r cannot both be 0 at the same time; and

[1090] (ii) s, t and v cannot all be 0 at the same time;

[1091] Z is an oligonucleoside moiety;

[1092] And, where appropriate, deprotection of the ligand and / or annealing of the second strand of the oligonucleoside moiety is carried out.

[1093] 69. The method according to item 68, wherein the compound of formula (XII) is prepared by reacting compounds of formula (XIV) and (XV):

[1094]

[1095] Each occurrence of R1 is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1096] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro;

[1097] Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen and sulfur;

[1098] q, r, s, t, and v are independently integers from 0 to 4, provided that:

[1099] (i) both q and r cannot be 0 simultaneously; and

[1100] (ii) all of s, t, and v cannot be 0 simultaneously;

[1101] Z is an oligonucleotide moiety.

[1102] 70. A method according to item 68 for preparing a compound according to any one of items 20, 25, 27, 29, 54, 56, and / or a composition according to any one of items 30, 31, 57, 58, wherein:

[1103] The compound of formula (XII) is of formula (XIIa):

[1104]

[1105] And the compound of formula (XIII) is of formula (XIIIa):

[1106]

[1107] Wherein the oligonucleotide comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1108] 71. A method according to item 68 for preparing a compound according to any one of items 20, 25, 28, 29, 55, 56, and / or a composition according to any one of items 30, 31, 57, 58, wherein:

[1109] The compound of formula (XII) is of formula (XIIb):

[1110]

[1111] And the compound of formula (XIII) is of formula (XIIIa):

[1112]

[1113] Wherein the oligonucleotide comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1114] 72. A method according to item 68, for preparing a compound according to any one of items 21, 26, 32, 34, 59, 61, and / or a composition according to any one of items 35, 36, 62, 63, wherein:

[1115] The compound of formula (XII) is of formula (XIIc):

[1116]

[1117] And the compound of formula (XIII) is of formula (XIIIa):

[1118]

[1119] Wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1120] 73. A method according to item 68, for preparing a compound according to any one of items 21, 26, 33, 34, 60, 61, and / or a composition according to any one of items 35, 36, 62, 63, wherein:

[1121] The compound of formula (XII) is of formula (XIId):

[1122]

[1123] And the compound of formula (XIII) is of formula (XIIIa):

[1124]

[1125] Wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1126] 74. A method according to any one of items 70 to 73, wherein:

[1127] The compound of formula (XIIIa) is of formula (XIIIb):

[1128]

[1129] 75. The method according to item 69, which depends on items 70 to 73, wherein:

[1130] The compound of formula (XIV) is of formula (XIVa) or formula (XIVb):

[1131]

[1132] And the compound of formula (XV) is of formula (XVa) or formula (XIVb):

[1133]

[1134] Wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein (i) the RNA duplex is linked at the 5' end of its second strand to an adjacent phosphate group in formula (XVa), or (ii) the RNA duplex is linked at the 3' end of its second strand to an adjacent phosphate group in formula (XVb).

[1135] 76. The compound of formula (XII):

[1136]

[1137] Wherein:

[1138] R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl each time it appears;

[1139] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen, and nitro;

[1140] X1 and X2 are independently selected from the group consisting of methylene, oxygen, and sulfur each time they appear;

[1141] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1142] (i) q and r cannot both be 0 at the same time; and

[1143] (ii) s, t, and v cannot all be 0 at the same time;

[1144] Z is an oligonucleoside moiety.

[1145] 77. The compound of formula (XIIa):

[1146]

[1147] 78. The compound of formula (XIIb):

[1148]

[1149] 79. Compounds of formula (XIIc):

[1150]

[1151] 80. Compounds of formula (XIId):

[1152]

[1153] 81. Compounds of formula (XIII):

[1154]

[1155] Wherein:

[1156] R1 is independently selected from the group consisting of hydrogen, methyl and ethyl each time it appears;

[1157] m is an integer from 1 to 6;

[1158] n is an integer from 1 to 10.

[1159] 82. Compounds of formula (XIIIa):

[1160]

[1161] 83. Compounds of formula (XIIIb):

[1162]

[1163] 84. Compounds of formula (XIV):

[1164]

[1165] Wherein:

[1166] R1 is selected from the group consisting of hydrogen, methyl and ethyl;

[1167] R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro;

[1168] X2 is selected from the group consisting of methylene, oxygen and sulfur;

[1169] s, t, v are independently integers from 0 to 4, provided that s, t and v cannot all be 0 at the same time.

[1170] 85. Compounds of formula (XIVa):

[1171]

[1172] 86. Compound of formula (XIVb):

[1173]

[1174] 87. Compound of formula (XV):

[1175]

[1176] Wherein:

[1177] R1, each occurrence independently, is selected from the group consisting of hydrogen, methyl and ethyl;

[1178] X1 is selected from the group consisting of methylene, oxygen and sulfur;

[1179] q and r are independently integers from 0 to 4, provided that: q and r cannot both be 0;

[1180] Z is an oligonucleoside moiety.

[1181] 88. Compound of formula (XVa):

[1182]

[1183] 89. Compound of formula (XVb):

[1184]

[1185] 90. Use of a compound according to any one of items 76, 81 to 84, 87 in the preparation of a compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63.

[1186] 91. Use of the compound of item 85 in the preparation of a compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2 = F.

[1187] 92. Use of the compound of item 86 in the preparation of a compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2 = OH.

[1188] Use of the compound of item 77 in the preparation of the compound of any one of items 20, 25, 27, 29, 54, 56, and / or the composition of any one of items 30, 31, 57, 58.

[1189] 94. Use of the compound of item 78 in the preparation of the compound of any one of items 20, 25, 28, 29, 55, 56, and / or the composition of any one of items 30, 31, 57, 58.

[1190] 95. Use of the compound of item 79 in the preparation of the compound of any one of items 21, 26, 32, 34, 59, 61, and / or the composition of any one of items 35, 36, 62, 63.

[1191] 96. Use of the compound of item 80 in the preparation of the compound of any one of items 21, 26, 33, 34, 60, 61, and / or the composition of any one of items 35, 36, 62, 63.

[1192] 97. Use of the compound of item 88 in the preparation of the compound of any one of items 20, 25, 27 to 29, 54 to 56, and / or the composition of any one of items 30, 31, 57, 58.

[1193] 98. Use of the compound of item 89 in the preparation of the compound of any one of items 21, 26, 32 to 34, 59 to 61, and / or the composition of any one of items 35, 36, 62, 63.

[1194] 99. A compound or composition obtained or obtainable by a method according to any one of items 68 to 75.

[1195] 100. A pharmaceutical composition comprising a compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63, and a pharmaceutically acceptable carrier, diluent or excipient.

[1196] 101. A compound according to any one of items 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 - 67, and / or a composition according to any one of items 30, 31, 35, 36, 57, 58, 62 and 63, for use in therapy.

[1197] In another aspect, the present invention can be applied to the compounds, methods, compositions or uses of the following items numbered 1 - 56 (wherein any reference to a formula in an item refers only to those formulas defined within items 1 - 56, which are reproduced Figure 7 herein).

[1198] 1. A compound comprising the following structure:

[1199]

[1200] Wherein:

[1201] r and s are independently integers selected from 1 to 16; and

[1202] Z is an oligonucleotide moiety.

[1203] 2. The compound according to item 1, wherein s is an integer selected from 4 to 12.

[1204] 3. The compound according to item 2, wherein s is 6.

[1205] 4. The compound according to any one of items 1 to 3, wherein r is an integer selected from 4 to 14.

[1206] 5. The compound according to item 4, wherein r is 6.

[1207] 6. The compound according to item 4, wherein r is 12.

[1208] 7. The compound according to item 5, which depends on item 3.

[1209] 8. The compound according to item 6, which depends on item 3.

[1210] 9. The compound according to any one of items 1 to 8, wherein Z is:

[1211]

[1212] Wherein:

[1213] Z1, Z2, Z3, Z4 are independently oxygen or sulfur each time they appear; and one of the bonds between P and Z2 and between P and Z3 is a single bond and the other is a double bond.

[1214] 10. The compound according to any one of items 1 to 9, wherein the oligonucleotide is an RNA compound capable of regulating, preferably inhibiting, the expression of a target gene.

[1215] 11. The compound according to item 10, wherein the RNA compound comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end.

[1216] 12. The compound according to item 11, preferably also depending on items 3 and 6, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[1217] 13. The compound according to item 11, preferably also depending on items 3 and 5, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[1218] 14. The compound of formula (II*), preferably depending on item 12:

[1219]

[1220] 15. The compound of formula (III*), preferably depending on item 13:

[1221]

[1222] 16. The compound as defined in any one of items 1 to 15, wherein the oligonucleoside comprises an RNA duplex, and the RNA duplex further comprises one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[1223] 17. The compound according to item 16, wherein the modification is selected from: 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[1224] 18. The compound according to any one of items 1 to 17, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more ends.

[1225] 19. The compound according to item 18, wherein the one or more degradation protection moieties are not present at the ends of the oligonucleoside chain carrying the linker / ligand moiety, and / or wherein the one or more degradation protection moieties are selected from: phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein the inverted abasic nucleosides are present at the distal end of the same strand as the end carrying the linker / ligand moiety.

[1226] 20. The compound according to any one of items 1 to 19, wherein the ligand moiety described by formula (I*) in item 1 comprises one or more ligands.

[1227] 21. The compound according to item 20, wherein the ligand moiety described by formula (I*) in item 1 comprises one or more carbohydrate ligands.

[1228] 22. The compound according to item 21, wherein the one or more carbohydrates can be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[1229] 23. The compound according to item 22, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[1230] 24. A compound according to item 23, wherein the one or more saccharides comprise one or more N-acetyl-galactosamine moieties.

[1231] 25. A compound according to item 24, which comprises two or three N-acetylgalactosamine moieties.

[1232] 26. A compound according to any one of the preceding items, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[1233] 27. A compound according to item 26, wherein the one or more ligands are linked in a bi-antennary or tri-antennary branched configuration.

[1234] 28. A compound according to items 20 to 27, wherein the moiety described by formula (I*) in item 1:

[1235]

[1236] is any one of formula (IV*), (V*) or (VI*), preferably formula (IV*):

[1237]

[1238] wherein:

[1239] A I is hydrogen, or a suitable hydroxyl protecting group;

[1240] a is an integer of 2 or 3; and

[1241] b is an integer from 2 to 5; or

[1242]

[1243] wherein:

[1244] A I is hydrogen, or a suitable hydroxyl protecting group;

[1245] a is an integer of 2 or 3; and

[1246] c and d are independently integers from 1 to 6; or

[1247]

[1248] wherein:

[1249] A I is hydrogen, or a suitable hydroxyl protecting group;

[1250] a is an integer of 2 or 3; and

[1251] e is an integer from 2 to 10.

[1252] 29. A compound according to any one of items 1 to 28, wherein the moiety described by formula (I*) in item 1

[1253]

[1254] is of formula (VII*):

[1255]

[1256] wherein:

[1257] A I is hydrogen;

[1258] a is an integer of 2 or 3.

[1259] 30. A compound according to item 28 or 29, wherein a = 2.

[1260] 31. A compound according to item 28 or 29, wherein a = 3.

[1261] 32. A compound according to item 28, wherein b = 3.

[1262] 33. A compound of formula (VIII*):

[1263]

[1264] 34. A compound of formula (IX*):

[1265]

[1266] 35. A compound according to item 33 or 34, wherein the oligonucleoside comprises an RNA duplex, and the RNA duplex further comprises one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[1267] 36. A compound according to item 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[1268] 37. A compound according to any one of items 33 to 36, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more ends.

[1269] 38. A compound according to item 37, wherein the one or more degradation protection moieties are not present at the ends of the oligonucleoside chain carrying the linker / ligand moiety, and / or wherein the one or more degradation protection moieties are selected from: phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, wherein the inverted abasic nucleosides are present at the distal end of the same chain as the end carrying the linker / ligand moiety.

[1270] 39. The compound according to item 33, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1271] 40. The compound according to item 34, wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1272] 41. A method for preparing the compound according to any one of items 1 to 40, which comprises reacting compounds of formula (X*) and (XI*):

[1273]

[1274] wherein:

[1275] r and s are independently integers selected from 1 to 16; and

[1276] Z is an oligonucleoside moiety;

[1277] and, where appropriate, deprotecting the ligand and / or annealing the second strand of the oligonucleoside.

[1278] 42. The method according to item 41, for preparing the compound according to any one of items 6, 8 to 14, 16 to 33 and 35 to 40, wherein:

[1279] The compound of formula (X*) is of formula (Xa*):

[1280]

[1281] and the compound of formula (XI*) is of formula (XIa*):

[1282]

[1283] wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 5' end of its second strand.

[1284] 43. The method according to item 41, for preparing a compound according to any one of items 5, 7, 9 to 13, 15 to 32, and 34 to 40, wherein:

[1285] The compound of formula (X*) is of formula (Xb*):

[1286]

[1287] And the compound of formula (XI*) is of formula (XIa*):

[1288]

[1289] Wherein the oligonucleoside comprises an RNA duplex, the RNA duplex comprises a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate group at the 3' end of its second strand.

[1290] 44. The method according to item 42 or 43, wherein:

[1291] The compound of (XIa*) is of formula (XIb*):

[1292]

[1293] 45. A compound of formula (X*):

[1294]

[1295] Wherein:

[1296] r is independently an integer selected from 1 to 16; and

[1297] Z is an oligonucleoside moiety.

[1298] 46. A compound of formula (Xa*):

[1299]

[1300] 47. A compound of formula (Xb*):

[1301]

[1302] 48. A compound of formula (XI*):

[1303]

[1304] Wherein:

[1305] s is independently an integer selected from 1 to 16; and

[1306] Z is an oligonucleotide moiety.

[1307] 49. A compound of formula (XIa*):

[1308]

[1309] 50. A compound of formula (XIb*):

[1310]

[1311] 51. Use of a compound according to any one of items 45 and 48 to 50 in the preparation of a compound according to any one of items 1 to 40.

[1312] 52. Use of the compound of item 46 in the preparation of a compound according to any one of items 6, 8 to 14, 16 to 33 and 35 to 40.

[1313] 53. Use of the compound of item 47 in the preparation of a compound according to any one of items 5, 7, 9 to 13, 15 to 32 and 34 to 40.

[1314] 54. A compound or composition obtainable or obtained by a method according to any one of items 41 to 44.

[1315] 55. A pharmaceutical composition comprising a compound according to any one of items 1 to 40, and a pharmaceutically acceptable carrier, diluent or excipient.

[1316] 56. A compound according to any one of items 1 to 40 for use in therapy.

[1317] Examples

[1318] The present invention will be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and those skilled in the art can conceive of various modifications or changes therefrom, and such modifications or changes should all be included within the spirit and scope of the invention and the scope of the claims.

[1319] Example 1 - Target Identification

[1320] Background

[1321] Genome-wide association (GWAS) studies aim to discover statistical associations between genetic variations in chromosomal DNA (genotypes) and an individual's physical or functional characteristics (phenotypes). There are several types of genetic variations, among which the most commonly studied in GWAS are single nucleotide polymorphisms (SNPs), where a single nucleotide in the genomic background DNA sequence differs between individuals. These SNPs can lead to changes in gene or protein function. This can occur directly (in the coding region of a gene) or indirectly (through effects on gene regulation). The mapping between SNPs and genes is not always 1:1, but can be many-to-one or one-to-many. GWAS studies have been used in drug discovery to identify genes whose variations are associated (either positively or negatively) with the risk of developing a particular disease, such as type 2 diabetes or atherosclerosis; or genes associated with specific consequences caused by these diseases, such as stroke or myocardial infarction. Occasionally, when most of the risk is concentrated in a single variant or when multiple variants map to a single gene, identifying this gene can yield clinically useful drug targets. But this is the exception, not the rule.

[1322] For complex (multifactorial) diseases such as diabetes, the more typical result of GWAS analysis is a long list of genes that are evaluated as being associated with the disease under study, but each gene only carries a very small fraction of the risk. How to use these long lists of weakly associated genes to elucidate biological mechanisms and drive drug target discovery is the core problem being addressed. In addition, there is often considerable uncertainty in mapping potential weakly associated SNPs to their associated genes or genes, and the relationships between genes, so the underlying biology is opaque. In this context, identifying clinically viable drug targets is extremely challenging and often fails.

[1323] The inventors have developed a proprietary computational method that uses network analysis methods to analyze such "noisy" GWAS (and other "omics") gene lists (which can contain hundreds of weakly associated genes and many mapping errors) to identify the underlying biology driving complex disease risk and find drug targets that conventional methods cannot discover.

[1324] To achieve this goal, the inventors adopted a proprietary network analysis method that allows them to assign multiple genes to a smaller number of driving processes; and to mine influential drug targets from these processes.

[1325] As described above, this method utilizes information that is typically overlooked in standard analysis - the interactions between known and predicted (using proprietary methods) genes (and proteins), and the prediction of "hidden players" - other genes with which the GWAS (or other "omics") genome also interacts.

[1326] Method for Identifying Identification Process and Target

[1327] The first step is to assume that the dysfunctions associated with "disease" should not be viewed at the single gene level. Instead, each gene belongs to a set whose members cooperate in the coordination of interacting proteins. Network Module Networks give rise to biology Process and the dysfunctions at the level of such processes or network modules should be considered as drivers of risk.

[1328] Each of the individual proteins in the network affected by SNPs contributes a small part to the overall dysfunction of the network module that controls the biological process. Network modules can interact with each other, resulting in dysfunctions at a coarser tissue level.

[1329] To this end, after fine mapping, protein-coding genes are selected from the gene list. Using a database of all possible protein-protein interactions (derived from external experiments) that are internally curated and an internal "network construction" algorithm - a series of viable networks are generated, including the maximum number of proteins in the list and the minimum number of estimated additional "hidden player" proteins. The algorithm attempts to find the best way to connect the protein-coding genes, using paths constrained by protein-protein interaction (PPI) data and inputting missing proteins according to a "cost function".

[1330] This method captures the relationships between the protein-coding genes in the GWAS list and adds other proteins ("hidden players") that are computationally involved in the same process.

[1331] In this way, multiple small effects are integrated in one or more networks to produce a greater effect.

[1332] The second step is to assume that the connection patterns within such networks are crucial for determining the impact of gene dysfunctions. This information is usually not easily accessible and is often overlooked in conventional analyses.

[1333] Using the networks obtained in the first step, functional enrichment analysis is performed. Functional enrichment analysis is different from conventional methods because, in addition to overlap, it also incorporates information about the estimated hidden players and the connection patterns of the proteins. That is, the relationships between the protein-coding genes in the GWAS list and the "hidden players" are used to identify the pathways crucial for the network structure.

[1334] To this end, an internally curated pathway database is used to define the set of proteins associated with a specific biological process. These sets of proteins are then network-tested by measuring the "structural impact" of removing common proteins on the network and assigning an "impact value" depending on the specific wiring pattern of the network. Further statistical controls are performed to ensure that any biases in the statistical properties of the proteins in the GWAS set are controlled.

[1335] The third step is to assume that due to the combined errors and uncertainties outlined above, the list of genes associated with the dysregulated functions is incomplete; moreover, due to the potential severity of some key proteins, their mutations cannot be tolerated. Therefore, it is necessary to "impute" the missing parts.

[1336] Next, the pathways from the "network enrichment" analyzed above are plotted in two-dimensional space, where each pathway is represented by a point and the proximity of the points is a measure of pathway similarity (see Figure 8 ). The pathway data is enhanced by using a search algorithm and the techniques of the above PPI database to add additional members that are "nearby in network space" according to another cost function. This allows pathways that do not share many proteins but share "neighbors" to be compared. Unsupervised machine learning methods are used to cluster similar pathways into groups. The biological functions (processes related to risk) of such groups are determined through expert interpretation of pathway annotations and protein annotations.

[1337] Next, an internal proprietary database of protein-protein relationships including the "direction" of interaction is used to reconstruct a directed network model from a selected group of pathway proteomes representing biological processes related to disease risk. This direction information comes from a series of public and internal databases and is supplemented by inferred directions from natural language processing of scientific publication texts. A network construction algorithm using these information sources is used to construct a "directed" model of key biological processes.

[1338] Then proprietary analysis techniques are applied to the network model to identify pharmacologically viable targets from the network whose knockout will have a significant impact on the network and, in turn, on the biological function being modeled. The algorithm makes extensive use of direction information and hierarchical relationships to identify targets with a set of specific properties that will make the above targets good siRNA targets. The targets are then further screened by protein class and hepatocyte specificity as needed.

[1339] Identification of Key Processes and siRNA Drug Targets in Type 2 Diabetes

[1340] The inventors have created a network model of type 2 diabetes using network biology methods. The network model is designed to capture all the important proteins involved in the process and their connections, and importantly, the direction of information flow between protein pairs.

[1341] The inventors have analyzed these network models using proprietary analytical methods. These methods use directional information to capture key "target" properties such as whether a protein is an integrator of information, a key conduit of information to other parts of the network, an influencer of key proteins, and the extent to which an influencer is influenced or influences other proteins (based on the absolute and relative quantities and directions of inputs and outputs). Directional information can also infer hierarchical relationships between proteins. Proteins with higher levels and certain properties can be superior to other proteins with other similar characteristics. The relative specificity and magnitude of each property relative to other properties enable the inventors to score and rank proteins according to their target suitability.

[1342] The ability to characterize the properties of these targets according to network relationships enables us to make judgments about selectivity and degree of influence in a selected context and, thus, the suitability for each given indication.

[1343] This enables the inventors to identify targets that provide improved treatment for type 2 diabetes. The analysis for this purpose is customized specifically for finding new and non-obvious targets, the knockout of which by GalNAc-siRNA in hepatocytes would be beneficial for the treatment of diabetes.

[1344] For this purpose, the inventors utilized a large GWAS meta-analysis of 898,930 individuals, 9% of whom were diabetic patients (Mahajan et al., Nature Genetics, 2018, 50, pp. 1505-1513).

[1345] Based on the GWAS meta-analysis, the inventors listed 257 genes, which were derived from 403 different association signals and were weakly associated with the risk of developing type 2 diabetes. The 257 genes were subdivided into Figure 9 the categories in.

[1346] Using the proprietary network analysis method described above, the inventors were able to identify a specific biological process: "post-translational modification of glycosylation", which is significantly associated with the risk of type 2 diabetes in normal and obese individuals. Standard "functional enrichment" methods were unable to identify this process and were not recognized by the authors of the meta-analysis (Mahajan et al., Nature Genetics, 2018, 50, pp. 1505-1513).

[1347] The inventors were also able to use their network-aware technology to demonstrate the restoration of known diabetes risk-related processes and to demonstrate an increased sensitivity of this method - see Figure 10 .

[1348] The inventors used multiple proprietary methods to reconstruct the network model of the process and used their analysis to rank individual hepatocyte genes based on predicted pharmacological effects and fitness for GalNAc-mediated siRNA knockdown to identify key target genes.

[1349] This approach enabled the inventors to identify three hepatocyte-expressed genes encoding secretase products, with B4GALT1 being the highest-ranked hepatocyte-expressed target in the analysis. While many proteins ranked in the upper quartile, only three proteins also passed the selection criteria of hepatocyte expression, secretion, and being an enzyme ( Figure 11 ).

[1350] Example 2: Synthesis of Tether 1

[1351] General Experimental Conditions:

[1352] Thin-layer chromatography (TLC) was performed using a 254 nm fluorescent indicator from Macherey-Nagel on an aluminum plate coated with silica. Compounds were visualized under ultraviolet light (254 nm) or after spraying with methanol (MeOH) or 5% H2SO4 in ninhydrin reagent according to Stahl (from Sigma-Aldrich) and then heating. Flash chromatography was performed using a Biotage Isolera One flash chromatography instrument equipped with a bivariate UV wavelength detector (200 - 400 nm) using Biotage Silica 10, 25, 50, or 100 g columns (Uppsala, Sweden).

[1353] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK Scientific.

[1354] An Acquity UPLC Protein BEH C4 column from Waters was used ( (1.7 μm, 2.1 x 100 mm), HPLC / ESI-MS was performed at 60 °C on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A (H2O containing 0.1% formic acid) and solvent B (acetonitrile (ACN) containing 0.1% formic acid). A gradient of 5 - 100% B was used at a flow rate of 0.4 mL / min over 15 minutes. Detector and conditions: Corona charged aerosol detection (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[1355] At room temperature on a Varian spectrometer at 500 MHz ( 1 1H NMR) and 125 MHz ( 13 13C NMR) were recorded 1 1H and 13 13C NMR spectra. Chemical shifts are in ppm, referenced to the solvent residual peaks (CDCl3 – 1 1H NMR: δ at 7.26 ppm and 13 13C NMR at 77.2 ppm; DMSO-d6 – 1 1H NMR: δ at 2.50 ppm and 13 13C NMR at 39.5 ppm). Coupling constants are in Hz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[1356] Synthesis Route of Conjugate Molecular Building Block TriGalNAc Tether 1:

[1357]

[1358] Preparation of Compound 2: Under argon, D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL), and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 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, which was purified by flash chromatography (gradient elution: 0 - 10% MeOH in 10 CV of DCM). A colorless oil product was obtained (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1359]

[1360] Preparation of Compound 4: Under argon, dissolve Compound 2 (2.30 g, 6.98 mmol, 1.0 eq.) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq.) in anhydrous DCM (40 mL), and add molecular sieves (5 g) to the solution. Stir the mixture at room temperature for 1 hour. Then add TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) to the mixture and stir the reaction overnight. Filter the molecular sieves, dilute the filtrate with DCM (100 mL) and wash with cold saturated aqueous NaHCO3 solution (100 mL) and water (100 mL). Separate the organic layer, dry it over Na2SO4 and remove the solvent under reduced pressure. Purify the crude material by flash chromatography (gradient elution: 0 - 3% MeOH in 10 CV of DCM) to obtain the title compound as a pale yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: Calculated for C 20 H 32 N4O 11 is 504.21. Found is 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).

[1361]

[1362] Preparation of Compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq.) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL, 1:1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed by vacuum / argon cycling (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to give the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). The compound was used without further purification. MS: Calculated for C 20 H 34 N2O 11 is 478.2. Found 479.4.

[1363]

[1364] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM / water (40 mL, 1:1 v / v), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 eq.) was added with vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure, and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% EtOAc in cyclohexane in 12 CV) to give the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (cyclohexane with 10% EtOAc)). MS: Calculated for C 33 H 53 NO 11 is 639.3. Found 640.9. 1 H NMR (500 MHz, DMSO-d6) δ 7.38 - 7.26 (m, 5H), 4.97 (s, 2H), 3.54 (t, 6H), 3.50 (s, 6H), 2.38 (t, 6H), 1.39 (s, 27H). 1313C 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).

[1365]

[1366] Preparation of Compound 8: Under argon, Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL), trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to obtain the compound in the form of the TFA salt (0.183 g, 98%). The compound was used without further purification. MS: Calculated for C 21 H 29 NO 11 is 471.6. Found 472.4.

[1367]

[1368] Preparation of Compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 eq.) 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 eq.), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution, and the reaction was stirred for 72 hours. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL), and washed with saturated aqueous NaHCO3 (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in 14 CV of DCM). A pale yellow oily product was obtained (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: Calculated for C 81 H 125 N7O41 It is 1852.9. The measured value is 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).

[1369]

[1370] Preparation of Compound 10: Dissolve the tritopic GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) in MeOH (15 mL), add 3 drops of acetic acid (AcOH) and Pd / C (30 mg). Degas the reaction mixture by vacuum / argon cycling (3x) and hydrogenate overnight under balloon pressure. After completion of the reaction, perform mass spectrometry analysis and filter the resulting mixture through a thin pad of diatomaceous earth. Evaporate the solvent and dry the obtained residue under high vacuum and use it for the next step without further purification. Obtain a pale yellow oily product (0.24 g, quantitative yield). MS: Calculated for C 73 H 119 N7O 39 It is 1718.8. The measured value is 1719.3.

[1371]

[1372] Preparation of Compound 11: Commercially available disuccinimidyl suberate (3.67 g, 9.9 mmol, 1.0 eq.) was dissolved in DMF (5 mL), and triethylamine (1.2 mL) was added. A solution of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0 eq.) in DMF (5 mL) was added dropwise to this solution. The reaction was stirred at room temperature for 3 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in 16 CV of DCM). A white solid product was obtained (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM)). MS: Calculated for C 15 H 23 N5O5 is 353.4. Found 354.3.

[1373]

[1374] Preparation of TriGalNAc(12): Under argon, the trivalent GalNAc compound 10 (0.35 g, 0.24 mmol, 1.0 eq.) and compound 11 (0.11 g, 0.31 mmol, 1.5 eq.) were dissolved in DCM (5 mL), and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure, the residue was dissolved in EtOAc (100 mL), and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was evaporated and the resulting crude material was purified by flash chromatography (elution gradient: 0 - 10% MeOH in 20 CV of DCM), to give the white fluffy solid title compound (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)). MS: Calculated for C 84 H 137 N 11 O 41 is 1957.1. Found 1959.6.

[1375] Conjugation of Tether 1 with siRNA Strand: Monofluoro Cyclooctyne (MFCO) Conjugation at the 5' or 3' End

[1376] 5'-end MFCO conjugation

[1377]

[1378] 3'-end MFCO conjugation

[1379]

[1380] General conditions for MFCO conjugation: The amine-modified single strand was dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer (pH 9.6 / dimethyl sulfoxide (DMSO) 4:6 (v / v)), and a solution of one molar equivalent of 35 mM MFCO-C6-NHS ester (Berry & Associates, catalog number LK 4300) in DMF was added to this solution. The reaction was carried out at room temperature, and after 1 hour, another molar equivalent of the MFCO solution was added. The reaction was allowed to proceed for another hour and monitored by LC / MS. At least two molar equivalents excess of the MFCO NHS ester reagent relative to the amino-modified oligonucleotide was required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered through a 1.2 μm filter from Sartorius, and then purified by reverse phase (RP HPLC) on a Pure instrument (GE Healthcare).

[1381] Purification was carried out using a Waters XBridge C18 Prep 19x 50 mm column. Buffer A was 100 mM TEAAc pH 7, and buffer B was 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60 °C. The UV trace was recorded at 280 nm. A gradient of 0 - 100% B was employed within 60 column volumes.

[1382] The fractions containing the full-length conjugated oligonucleotide were combined, precipitated in the refrigerator with 3M NaOAc, pH 5.2 and 85% ethanol, and the collected pellet was dissolved in water. The sample was desalted by size exclusion chromatography and concentrated using a speed-vac concentrator to obtain the conjugated oligonucleotide with a separation yield of 40 - 80%.

[1383] 5’-GalNAc-T1 conjugate

[1384]

[1385] 3’-GalNAc-T1 conjugate

[1386]

[1387] General procedure for TriGalNAc conjugation: The MFCO-modified single strand was dissolved in water at 2000 OD / mL, and then a solution of one equivalent of compound 12 (10 mM) in DMF was added to this solution. The reaction was carried out at room temperature. After 3 hours, a 0.7 molar equivalent solution of compound 12 was added. The reaction was carried out overnight and monitored by LCMS for completion. The conjugate was diluted 15-fold in water, filtered through a 1.2 μm filter from Sartorius, and then purified by RP-HPLC on a Pure instrument (GE Healthcare).

[1388] RP-HPLC purification was carried out using a Waters XBridge C18 Prep 19x 50 mm column. Buffer A was 100 mM triethylammonium acetate (pH 7), and buffer B was 95% acetonitrile in buffer A. The flow rate was 10 mL / min, and the temperature was 60 °C. The UV trace was recorded at 280 nm. A gradient of 0-100% B was employed within 60 column volumes.

[1389] The fractions containing the full-length conjugated oligonucleotide were combined, precipitated in the refrigerator with 3M NaOAc, pH 5.2 and 85% ethanol, and the collected precipitate was dissolved in water to obtain an oligonucleotide solution of approximately 1000 OD / mL. The O-acetates were removed by adding 20% ammonia. The quantitative removal of these protecting groups was verified by LC-MS.

[1390] The conjugate was desalted by size exclusion chromatography using Sephadex G25 fine resin (GE Healthcare) on a Pure (GE Healthcare) instrument to obtain the conjugated oligonucleotide with a separation yield of 50-70%.

[1391] The following protocol further lists the synthetic route:

[1392] Scheme 1:

[1393]

[1394] Scheme 2:

[1395]

[1396] Scheme 3:

[1397]

[1398] Scheme 4:

[1399]

[1400] Scheme 5:

[1401]

[1402] Example 3: Duplex Annealing

[1403] To generate the desired siRNA duplexes, two complementary strands were annealed by combining equimolar aqueous solutions of the two strands. The mixture was placed in a 70 °C water bath for 5 minutes and then cooled to ambient temperature over 2 hours. The duplexes were lyophilized for 2 days and stored at -20 °C.

[1404] On a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system, the duplexes were analyzed by analytical SEC HPLC on a Superdex TM 75 Increase 5 / 150GL column 5 x 153 - 158 mm (Cytiva). The mobile phase consisted of 1x PBS containing 10% acetonitrile. At room temperature, an isocratic gradient was run at a flow rate of 1.5 mL / min for 10 min. UV traces were recorded at 260 and 280 nm. Water (LC-MS grade) was purchased from Sigma-Aldrich and phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[1405] Example 4: Synthesis of Tether 2

[1406] General experimental conditions:

[1407] Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were visualized under ultraviolet light (254 nm) or after spraying with methanol (MeOH) or 5% H2SO4 in ninhydrin reagent according to Stahl (from Sigma-Aldrich) and then heating. Flash chromatography was performed using a Biotage Isolera One flash chromatography instrument equipped with a dual-variable UV wavelength detector (200 - 400 nm) using Biotage Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1408] All wet chemical reactions were carried out using dry glassware, anhydrous solvents, and an argon atmosphere under anhydrous conditions. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH+Co.KG. D-galactosamine pentaacetate was purchased from AK Scientific.

[1409] HPLC / ESI-MS was performed on a Waters Acquity UPLC Protein BEH C4 column ( 1.7 μm, 2.1 x 100 mm) at 60 °C on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A (H2O containing 0.1% formic acid) and solvent B (acetonitrile (ACN) containing 0.1% formic acid). A gradient of 5 - 100% B was used at a flow rate of 0.4 mL / min over 15 minutes. Detector and conditions: Corona charged aerosol detection (from ESA). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[1410] 1H and 1 13C NMR spectra were recorded at room temperature on a Varian spectrometer at 500 MHz ( 13 1H NMR) and 125 MHz ( 1 13C NMR). Chemical shifts are reported in ppm, referenced to solvent residual peaks (CDCl3 – 13 1H NMR: δ at 7.26 ppm and 1 13C NMR at 77.2 ppm; DMSO-d6 – 13 1H NMR: δ at 2.50 ppm and 1 13C NMR at 39.5 ppm). Coupling constants are reported in Hz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m). 13 13C NMR). Coupling constants are reported in Hz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[1411] Synthetic route of the conjugate molecular building block TriGalNAc linker 2:

[1412]

[1413] Preparation of Compound 2: Under argon, D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL), and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 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, which was purified by flash chromatography (gradient elution: 0 - 10% MeOH in 10 CV of DCM). A colorless oil product was obtained (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1414]

[1415] Preparation of Compound 4: Under argon, Compound 2 (2.30 g, 6.98 mmol, 1.0 eq.) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq.) were dissolved in anhydrous DCM (40 mL), and molecular sieves (5 g) were added to the solution. The mixture was stirred at room temperature for 1 hour. Then TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was added to the mixture and the reaction was stirred overnight. The molecular sieves were filtered off, 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 10 CV of DCM) to give the title compound as a pale yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: Calculated for C 20 H 32 N4O 11 is 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). 1313C 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).

[1416]

[1417] Preparation of Compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq.) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL, 1:1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed by vacuum / argon cycling (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to give the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). This compound was used without further purification. MS: Calculated for C 20 H 34 N2O 11 is 478.2. Found 479.4.

[1418]

[1419] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM / water (40 mL, 1:1 v / v), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 eq.) was added with vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure, and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% EtOAc in cyclohexane in 12 CV) to give the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (cyclohexane with 10% EtOAc)). MS: Calculated for C 33 H 53 NO 11 is 639.3. Found 640.9. 11H 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 13C 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).

[1420]

[1421] Preparation of Compound 8: Under argon, Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL), trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to give the compound in the form of the TFA salt (0.183 g, 98%). The compound was used without further purification. MS: Calculated for C 21 H 29 NO 11 is 471.6. Found value is 472.4.

[1422]

[1423] Preparation of Compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 eq.) 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 eq.), 1-hydroxybenzotriazole hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution, and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL), and washed with saturated aqueous NaHCO3 solution (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated and the crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in 14 CV of DCM). A pale yellow oily product was obtained (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: Calculated for C 81 H 125 N7O 41 was 1852.9. Found 1854.7. 1 H NMR (500 MHz, DMSO-d6) δ 7.90 - 7.80 (m, 10H), 7.65 - 7.62 (m, 4H), 7.47 - 7.43 (m, 3H), 7.38 - 7.32 (m, 8H), 5.24 - 5.22 (m, 3H), 5.02 - 4.97 (m, 4H), 4.60 - 4.57 (m, 3H), 4.07 - 3.90 (m 10H), 3.67 - 3.36 (m, 70H), 3.23 - 3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80 - 1.78 (m, 17H). 1313C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[1424]

[1425] Preparation of Compound 10: The tris-antennary GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) 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 (3x) and hydrogenated overnight under balloon pressure. After completion of the reaction, mass spectrometry was performed, and the resulting mixture was filtered through a thin pad of diatomaceous earth. The solvent was evaporated and the obtained residue was dried under high vacuum and used for the next step without further purification. A pale yellow oily product (0.24 g, quantitative yield) was obtained. MS: Calculated for C 73 H 119 N7O 39 is 1718.8. Found 1719.3.

[1426]

[1427] Preparation of Compound 14: Under argon, the tris-antennary GalNAc compound 10 (0.45 g, 0.26 mmol, 1.0 eq.), HBTU (0.19 g, 0.53 mmol, 2.0 eq.) and DIPEA (0.23 mL, 1.3 mmol, 5.0 eq.) were dissolved in DCM (10 mL). A solution of compound 13 (0.14 g, 0.53 mmol, 2.0 eq.) in DCM (5 mL) was added dropwise to this mixture. The reaction was stirred at room temperature overnight. The solvent was removed, the residue was dissolved in EtOAc (50 mL), washed with water (50 mL) and dried over Na2SO4. The solvent was evaporated and the crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM for 20 CV). A white fluffy solid product (0.25 g, 48%, rf = 0.4 (10% MeOH in DCM)) was obtained. MS: Calculated for C88 H 137 N7O 42 It is 1965.1. The measured value is 1965.6.

[1428]

[1429] Preparation of TriGalNAc(15): Dissolve the trisialyl GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) in EtOAc (15 mL), and add Pd / C (40 mg). Degas the reaction mixture using a vacuum / argon cycle (3x), and hydrogenate overnight under balloon pressure. Monitor the completion of the reaction by mass spectrometry, and filter the resulting mixture through a thin pad of diatomaceous earth. Remove the solvent under reduced pressure and dry the resulting residue under high vacuum overnight. The residue can be used for conjugation with oligonucleotides without further purification (0.28 g, quantitative yield). MS: Calculated for C 81 H 131 N7O 42 It is 1874.9. The measured value is 1875.3.

[1430] Conjugation of Linker 2 with siRNA strand: TriGalNAc Linker 2 (GalNAc-T2) is conjugated at the 5'-end or 3'-end

[1431] 5'-GalNAc-T2 conjugate

[1432]

[1433] 3'-GalNAc-T2 conjugate

[1434]

[1435] Preparation of TriGalNAc linker 2 NHS ester: Add N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol) to a solution of the carboxylic acid linker 2 (compound 15, 227 mg, 121 μmol) in DMF (2.1 mL). Stir the solution at room temperature for 18 hours and use it for the subsequent conjugation reaction without purification.

[1436] General procedure for conjugation of triGalNAc linker 2: The amine-modified single strand was dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer (pH 9.6 / DMSO 4:6 (v / v)), and then a solution of one molar equivalent of linker 2 NHS ester (57 mM) in DMF was added to this solution. The reaction was carried out at room temperature, and after 1 hour, another molar equivalent of the NHS ester solution was added. The reaction was allowed to proceed for another hour, and the progress of the reaction was monitored by LCMS. At least two molar equivalents of excess NHS ester reagent relative to the amino-modified oligonucleotide were required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered once through a 1.2 μm filter from Sartorius, and then purified by reverse-phase RP HPLC on a Pure (GE Healthcare) instrument.

[1437] Purification was carried out using a Waters XBridge C18 Prep 19x 50 mm column. Buffer A was 100 mM TEAA pH 7, and buffer B contained 95% acetonitrile in buffer A. The flow rate was 10 mL / min, and the temperature was 60 °C. The UV trace at 280 nm was recorded. A gradient of 0 - 100% B was employed within 60 column volumes.

[1438] The fractions containing the full-length conjugated oligonucleotide were combined, precipitated in the refrigerator with 3M NaOAc, pH 5.2 and 85% ethanol, and then dissolved in water at 1000 OD / mL. O-acetates were removed with 20% aqueous ammonium hydroxide until completion (monitored by LC-MS).

[1439] The conjugate was desalted by size exclusion chromatography on a Pure (GE Healthcare) instrument using Sephadex G25 fine resin to obtain the conjugated oligonucleotide with a separation yield of 60 - 80%.

[1440] The conjugate was characterized by HPLC-MS analysis using a 2.1 x 50 mm XBridge C18 column (Waters) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics). Buffer A was a solution of 16.3 mM triethylamine, 100 mM HFIP in H2O with 1% MeOH, and buffer B contained 95% MeOH in buffer A. The flow rate was 250 μL / min and the temperature was 60 °C. UV traces at 260 and 280 nm were recorded. A gradient of 1 - 100% B was employed over 31 minutes.

[1441] The following protocol further lists the synthetic route:

[1442] Scheme 6:

[1443]

[1444] Scheme 7:

[1445]

[1446] Scheme 8:

[1447]

[1448] Scheme 9:

[1449]

[1450] Example 5: Duplex annealing

[1451] To generate the desired siRNA duplex, two complementary strands were annealed by combining equimolar aqueous solutions of the two strands. The mixture was placed in a 70 °C water bath for 5 minutes and then cooled to ambient temperature over 2 hours. The duplex was lyophilized for 2 days and stored at -20 °C.

[1452] On a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system, via Superdex TMAnalyze the duplex by SEC HPLC using a 75 Increase 5 / 150GL column (5 x 153 - 158 mm, Cytiva). The mobile phase consists of 1x PBS containing 10% acetonitrile. Run an isocratic gradient at a flow rate of 1.5 mL / min for 10 min at room temperature. Record the UV traces at 260 and 280 nm. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[1453] Example 6: Alternative synthetic route for the conjugate component TriGalNAc-Linker 2

[1454]

[1455]

[1456] Conjugation of Linker 2 with siRNA strand: TriGalNAc Linker 2 (GalNAc-T2) is conjugated at the 5'-end or 3'-end

[1457] Conjugation conditions

[1458]

[1459] Pre-activation: At 25 °C, add TFA-O-PFP (15 μl, 21 eq.) to a solution of compound 15 (16 μmol, 4 eq.) in DMF (160 μL), then add DIPEA (23 μl, 32 eq.). Shake the tube at 25 °C for 2 h. Quench the reaction with H2O (10 μL).

[1460] Coupling: Dilute the resulting mixture with DMF (400 μl), then add the oligoamine solution (4.0 μmol in 10x PBS, pH 7.4, 500 μL; final oligoamine (oligo) concentration in the organic and aqueous solutions: 4 μmol / ml = 4 mM). Shake the tube at 25 °C for 16 h and analyze the reaction by LCMS. Treat the resulting mixture with 28% NH4OH (4.5 ml) and shake at 25 °C for 2 h. Analyze the mixture by LCMS, concentrate, and purify by IP-RP HPLC to produce the oligonucleotide conjugated to the tether 2 GalNAc. 5'-GalNAc-T2 conjugate

[1461]

[1462] 3'-GalNAc-T2 conjugate

[1463]

[1464] Example 7: Solid-phase synthesis method: scale ≤ 1 mmol

[1465] The synthesis of the sense and antisense strands of siRNA was carried out on a MerMade 192X synthesizer, which has a commercially available solid support made of controlled pore glass with a universal linker (universal CPG, loading of 40 μmol / g; LGC Biosearch or Glen Research).

[1466] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[1467] The 2'-O-methyl phosphoramidites used were as follows: 5'-(4,4'-dimethoxytriphenylmethyl)-N-benzoyl-adenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetyl-cytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytriphenylmethyl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytriphenylmethyl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1468] The 2'-F phosphoramidites used were as follows: 5'-dimethoxytriphenylmethyl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytriphenylmethyl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytriphenylmethyl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytriphenylmethyl-deoxythymidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1469] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphoramidite which was dissolved in DMF / MeCN (1:4, v / v). 0.02 M iodine in acetonitrile / pyridine / H2O (DNAchem) was used as the oxidation reagent. 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v was used for thiolation of the phosphorothioate bond. 0.25 M mM 5-ethylthiotetrazole (ETT) in acetonitrile was used as the activator solution.

[1470] Inverted nucleoside phosphoramidites, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[1471] In each cycle, DMT was removed using a deblocking solution (3% TCA in DCM, (DNAchem)).

[1472] The coupling time was 180 seconds. The oxidizer contact time was set to 80 seconds and the thiolation time was 2 * 100 seconds.

[1473] At the end of the synthesis, the oligonucleotide was cleaved from the solid support using an NH4OH:EtOH solution 4:1 (v / v) (TCI) at 45 °C for 20 hours. The solid support was then filtered off, the filter was thoroughly washed with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[1474] The oligonucleotide was treated using an Amicon Ultra-2 centrifugal filtration device, PBS buffer (10x, Teknova, pH 7.4, sterile) by ultracentrifugation, or by EtOH precipitation with 1M sodium acetate to form the sodium salt.

[1475] Single-strand identity was evaluated by MS ESI-, then annealed in water to form the final double-stranded siRNA, and duplex purity was evaluated by size exclusion chromatography.

[1476] Example 8: Solid-phase synthesis method: scale ≥ 5 mmol

[1477] The synthesis of the sense and antisense strands of siRNA was carried out on a MerMade12 synthesizer with a commercially available solid support made of controlled pore glass with a universal linker (universal CPG, loading 40 μmol / g; LGC Biosearch or Glen Research), Scale at 5 μmol. At 12 μmol on a 3'-PT-amino modifier C6 CPG The sense strand for 3'-conjugation was synthesized on a solid support with a loading of 86 μmol / g (LGC).

[1478] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[1479] 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.

[1480] 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.

[1481] 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).

[1482] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphoramidite which was dissolved in DMF / MeCN (1:4, v / v). 0.02 M iodine in acetonitrile / pyridine / H2O (DNAchem) was used as the oxidation reagent. 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v was used for thiolation of the phosphorothioate bond. 0.25 M mM 5-ethylthiotetrazole (ETT) in acetonitrile was used as the activator solution.

[1483] In each cycle, DMT was removed using a deblocking solution (3% TCA in DCM, (DNAchem)).

[1484] For the strands synthesized on universal CPG, coupling was carried out with 8 eq. of amidite for 130 seconds. The oxidation time was 47 seconds and the thiolation time was 210 seconds.

[1485] For the strands synthesized on 3'-PT-amino modifier C6 CPG, coupling was carried out with 8 eq. of amidite for 2 * 150 seconds. The oxidation time was 47 seconds and the thiolation time was 250 seconds.

[1486] At the end of synthesis, the oligonucleotides were cleaved from the solid support with NH4OH:EtOH solution 4:1 (v / v) (TCI) at 45 °C for 20 hours. Then the solid support was filtered off, the filter was thoroughly washed with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[1487] The oligonucleotides were treated by EtOH precipitation with 1 M sodium acetate to form the sodium salts.

[1488] Single-stranded oligonucleotides were purified by IP-RP HPLC on a Xbridge BEH C18 5 μm, 19 x 150 mm (Waters) column with a gradient increase of B in A. Mobile phase A: 240 mM HFIP, 7 mM TEA and 5% aqueous methanol; Mobile phase B: 240 mM HFIP, 7 mM TEA in methanol.

[1489] The single-stranded purity and identity were evaluated by UPLC / MS ESI- on a Xbridge BEH C18 2.5 μm, 3 x 50 mm (Waters) column with a gradient increase of B in A. Mobile phase A: 100 mM HFIP, 5 mM TEA in aqueous solution; Mobile phase B: 20% Mobile phase A: 80% acetonitrile (v / v).

[1490] The sense strands were conjugated according to the protocol provided in any one of Examples 2, 4, 6.

[1491] Then the sense strands and the antisense strands were annealed in water to form the final double-stranded siRNA, and the duplex purity was evaluated by size exclusion chromatography.

[1492] The present invention is not limited to the scope of the specifically disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods will become apparent from the description and teachings herein. Such changes can be made without departing from the true scope and spirit of the disclosure, and such changes are intended to fall within the scope of the disclosure.

[1493] Example 9: Pharmacological study of B4GALT1

[1494] ETX internal computational biology analysis identified B4GALT1 encoding β-1,4-galactosyltransferase 1, which is a gene associated with type 2 diabetes (T2D) (see Example 1). Here, we established B4GALT1 as a potential therapeutic target for type 2 diabetes. GalNAc-siRNA targeting mouse liver B4GALT1 designed by computer was synthesized and tested to verify the rationality of the following hypothesis: significant knockdown of liver B4GALT1 mRNA reduces the levels of plasma LDL-c, fibrinogen, and fasting blood glucose.

[1495] In vitro dose-response assay to screen for effective molecules

[1496] Through in vitro dose-response assays, gene knockdown in primary mouse hepatocytes (PMH) was measured to test 20 GalNAc-siRNAs targeting liver B4GALT1. Primary C57BL / 6 mouse hepatocytes (PMH) were freshly isolated by a two-step collagenase liver perfusion method. Cells were cultured in DMEM (Gibco - 11995 - 092) supplemented with FBS, penicillin / streptomycin, HEPES, and L-glutamine. Cells were cultured in a humidified incubator at 37 °C in an atmosphere of 5% CO2. Within 2 hours after isolation, PMH were seeded at a density of 36,000 cells / well in a conventional 96-well tissue culture plate. The dose-response analysis in PMH was performed by directly incubating the cells in a free uptake environment, where the final concentrations of GalNAc-siRNA were 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 nM. In the control wells, cells were incubated without the addition of GalNAc-siRNA. After 48 hours of incubation, the cells were harvested for RNA extraction. Total RNA was extracted using an RNeasy kit according to the manufacturer's instructions (Qiagen, Shanghai, China). After reverse transcription, real-time quantitative PCR was performed using an ABI Prism 7900HT to detect the relative abundance of B4GALT1 mRNA normalized to the housekeeping gene GAPDH, and the expression of the target gene in each test sample was determined by relative quantification using the comparative Ct (ΔΔCt) method. This method measures the Ct difference (ΔCt) between the target gene and the housekeeping gene. 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 with good activity were selected for EC using a 10-point concentration curve 50 determination ( Figure 12 ).

[1497] In Vivo Pharmacology of Four Selected GalNAc-siRNAs

[1498] The pharmacodynamic activities of four selected B4GALT1 GalNAc-siRNAs were measured in vivo. Twelve C57BL / 6 male mice were assigned for each of GalNAc-siRNA, ETXM619, ETXM624, ETXM628, and ETXM633. Five mice were assigned as untreated control group. On day 0 (defined as the date when the mice were first dosed), day 3, and day 7, the mice were dosed subcutaneously with ETXM (10 mg / kg). Three mice in each treatment group were sacrificed on day 3, day 7, day 10, and day 14. After termination, liver tissues and plasma samples were collected for further analysis. The samples on day 3 were used to evaluate the single-dose effect of ETXM administered on day 0. The samples on day 7 represented the repeated-dose effect of ETXM administered on day 0 and day 3. Similarly, the samples on day 10 and day 14 represented the repeated-dose effect of ETXM administered on day 0, day 3, and day 7. Five mice assigned as control group were sacrificed on day 14.

[1499] B4GALT1 gene knockdown in mouse liver

[1500] The collected liver samples were used to measure the B4GALT1 mRNA knockdown level by RT-qPCR. After collection, each tissue was treated with RNAlater and stored overnight at 4 °C, then stored at -80 °C until further analysis. The liver tissues were homogenized with TRIZOL for RNA extraction. The RNA samples were adjusted to 400 ng / μL and reverse transcribed into cDNA using the FastKing RT kit manufactured by TIANGEN Company. 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 It was shown that all test articles exhibited > 50% gene knockdown efficiency on day 3, 7, 10, and 14.

[1501] Terminal plasma collection and measurement of plasma biomarkers using a biochemical analyzer

[1502] Terminal plasma samples were collected via the submandibular vein after fasting for 4 - 5 hours. The blood samples were collected in heparin sodium-coated tubes and then centrifuged at 7,000 g for 10 min at 4 °C to obtain plasma samples. The plasma samples were used to measure AST, ALT, albumin, ALP, BUN, CREA, TBIL, glucose, total cholesterol, LDL-c, HDL-c, triglyceride, and NEFA (free fatty acids) by a biochemical analyzer.

[1503] Measurement of plasma insulin and fibrinogen levels using an ELISA kit

[1504] 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 levels were measured using a mouse insulin ELISA kit (Mercodia, 10-1247-01) according to the manufacturer's protocol. Fibrinogen plasma levels were measured using a mouse fibrinogen antigen assay kit (Innovative Research, IMSFBGKTT).

[1505] Effect of B4GALT1 gene silencing in biomarker regulation

[1506] Equality tests of the means of the untreated control group (n = 5) and the treatment group on day 14 (groups receiving subcutaneous administration of ETXM619, ETXM624, ETXM628, or ETXM633 on days 0, 3, and 7; n = 3 per group, total n = 12) were performed under the null hypothesis by two-tailed t-tests. Statistically significant differences were detected in the mean values of the efficacy biomarker readings, with an 18.8% decrease in LDL-C (p < 0.05); a 21.0% decrease in fasting glucose (p < 0.05); and a 29.6% decrease in fibrinogen (p < 0.01)( Figure 14 ).

Claims

1. An inhibitor of B4GALT1 expression and / or function for treating diabetes.

2. An inhibitor of post-translational glycosylation for treating diabetes, such as an inhibitor of B4GALT1 expression and / or function.

3. The inhibitor used according to claim 1 or 2, which is an siRNA oligomer, usually conjugated to one or more ligand moieties.

4. The inhibitor used according to claim 3, wherein the one or more ligand moieties comprise one or more GalNAc ligands and / or one or more GalNAc ligand derivatives.

5. An inhibitor of post-translational glycosylation, such as an inhibitor of B4GALT1 expression and / or function, wherein the inhibitor is conjugated to one or more ligand moieties.

6. The inhibitor according to claim 5, wherein the inhibitor comprises an siRNA oligomer conjugated to one or more ligand moieties.

7. The inhibitor according to claim 5 or 6, wherein the one or more ligand moieties comprise one or more GalNAc ligands.

8. The inhibitor according to any one of claims 5-7, wherein the one or more ligand moieties comprise one or more GalNAc ligand derivatives.

9. The inhibitor or the inhibitor used according to one or more of the foregoing claims, wherein the target of the inhibitor is selected from B4GALT1.

10. The inhibitor or the inhibitor used according to one or more of the foregoing claims, which is an siRNA oligomer having a first strand and a second strand, wherein: i) The length of the first strand of the siRNA ranges from 15 to 30 nucleotides, preferably from 19 to 25 nucleotides, more preferably 23 or 25 nucleotides; even more preferably 23 nucleotides; and / or ii) The length of the second strand of the siRNA ranges from 15 to 30 nucleotides, preferably from 19 to 25 nucleotides, more preferably 21 nucleotides.

11. The inhibitor or the inhibitor used according to claim 10, wherein the second sense strand further comprises one or more abasic nucleotides in the terminal region of the second strand, and wherein the one or more abasic nucleotides are linked to adjacent nucleotides by reverse internucleoside bonds.

12. The inhibitor or the inhibitor used according to claim 11, wherein the second strand comprises: i Two or more abasic nucleotides in the terminal region of the second strand; and / or ii Two or more abasic nucleotides in the 5' or 3' terminal region of the second strand; and / or iii Two or more abasic nucleotides in the 5' or 3' terminal region of the second strand, wherein the abasic nucleotides are present in the overhangs as described herein; and / or iv Two or more consecutive abasic nucleotides in the terminal region of the second strand, wherein preferably one of the abasic nucleotides is the terminal nucleotide; and / or v Two or more consecutive abasic nucleotides in the 5' or 3' terminal region of the second strand, wherein preferably one of the abasic nucleotides is the terminal nucleotide in the 5' or 3' terminal region of the second strand; and / or vi A reverse internucleoside bond links at least one abasic nucleoside to an adjacent base nucleoside in the terminal region of the second strand; and / or vii A reverse internucleoside bond links at least one abasic nucleoside to an adjacent base nucleoside in the 5’ or 3’ terminal region of the second strand; and / or viii An abasic nucleoside serves as the penultimate nucleoside, which is linked by a reverse bond to a nucleoside that is not the terminal nucleoside (referred to herein as the antepenultimate nucleoside); and / or ix When reading the strand in the direction towards the terminal, the abasic nucleoside serves as two terminal nucleosides linked by a 5’-3’ bond; x When reading the strand in the direction towards the terminal containing the terminal nucleoside, the abasic nucleoside serves as two terminal nucleosides linked by a 3’-5’ bond; xi An abasic nucleoside serves as the two terminal positions, wherein the penultimate nucleoside is linked to the antepenultimate nucleoside by the reverse bond, and wherein the reverse bond is a 5-5’ reverse bond or a 3’-3’ reverse bond; xii An abasic nucleoside serves as the two terminal positions, wherein the penultimate nucleoside is linked to the antepenultimate nucleoside by the reverse bond, and wherein (1) the reverse bond is a 5-5’ reverse bond, and when reading towards the terminal containing the terminal abasic nucleoside and the penultimate abasic nucleoside, the bond between the terminal abasic nucleoside and the penultimate abasic nucleoside is 3’5’; or (2) the reverse bond is a 3-3’ reverse bond, and when reading towards the terminal containing the terminal abasic nucleoside and the penultimate abasic nucleoside, the bond between the terminal abasic nucleoside and the penultimate abasic nucleoside is 5’3’.

13. The inhibitor or inhibitor used according to claim 11 or 12, wherein the reverse internucleoside bond is located in the terminal region distal to the 5’ terminal region of the second strand, or in the terminal region distal to the 3’ terminal region of the second strand.

14. The inhibitor or inhibitor used according to any one of claims 11 to 13, wherein the reverse internucleoside bond is a 3’3 reverse bond.

15. The inhibitor or inhibitor used according to any one of claims 11 to 13, wherein the reverse internucleoside bond is a 5’5 reverse bond.

16. The inhibitor or inhibitor used according to any one of claims 10 to 15, wherein one or more nucleosides of the first strand and / or the second strand are modified to form modified nucleosides.

17. The inhibitor or inhibitor used according to claim 16, wherein the modification is a modification at the 2’-OH group of the ribose, optionally selected from 2’-Me or 2’-F modifications.

18. The inhibitor or inhibitor used according to claim 16 or 17, wherein counting from position 1 of the first strand, the first strand contains 2’-F at any position among position 14, position 2, position 6, or any combination thereof.

19. The inhibitor or inhibitor used according to any one of claims 16 to 18, wherein, counting from position 1 of the second strand, the second strand comprises a 2'-F modification at position 7 and / or 9, and / or 11 and / or 13.

20. The inhibitor or inhibitor used according to any one of claims 16 to 19, wherein each of the first strand and the second strand comprises a 2'-Me and a 2'-F modification.

21. The inhibitor or inhibitor used according to any one of claims 16 to 20, which is an siRNA, wherein the siRNA comprises at least one heat-destabilizing modification, counting from position 1 of the first strand, which is suitably located at one or more positions from position 1 to 9 of the first strand, and / or suitably located at one or more 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.

22. The inhibitor or inhibitor used according to claim 21, wherein, counting from position 1 of the first strand, the siRNA comprises at least one heat-destabilizing modification at position 7 of the first strand.

23. The inhibitor or inhibitor used according to any one of claims 16 to 22, which is an siRNA, wherein, counting from position 1 of the second strand, the siRNA comprises 3 or more 2'-F modifications at positions 7 to 13 of the second strand, for example, 4, 5, 6 or 7 2'-F modifications at positions 7 to 13 of the second strand.

24. The inhibitor or inhibitor used according to any one of claims 16 to 23, which is an siRNA, wherein, counting from position 1 of the second strand, the second strand comprises at least 3, for example 4, 5 or 6 2'-Me modifications at positions 1 to 6 of the second strand.

25. The inhibitor or inhibitor used according to any one of claims 16 to 24, which is an siRNA, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3'-terminal region, preferably comprising the terminal nucleoside of the 3'-terminal region, or at least within 1 or 2 nucleosides of the terminal nucleoside of the 3'-terminal region.

26. The inhibitor or inhibitor used according to any one of claims 16 to 25, which is an siRNA, wherein the first strand comprises 7 consecutive 2'-Me modifications in the 3'-terminal region, preferably comprising the terminal nucleoside of the 3'-terminal region.

27. The inhibitor or inhibitor used according to any one of claims 16 to 22, which is an siRNA, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any of the following: (Me)8–(F)3–(Me) 10 。 28. The inhibitor or inhibitor used according to any one of claims 16 to 22 or 27, which is an siRNA, wherein the nucleosides of the first strand comprise a 2'-sugar modification pattern, wherein the modification is at least selected from 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications of the first strand does not consist of four or six 2'-F modifications.

29. The inhibitor or inhibitor used according to any one of claims 16 to 22 or 27 to 28, which is an siRNA, wherein the modification is at least selected from 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.

30. The inhibitor or inhibitor used according to any one of claims 16 to 29, wherein the siRNA oligomer further comprises one or more phosphorothioate internucleotide linkages.

31. The inhibitor or inhibitor used according to claim 30, wherein the one or more phosphorothioate internucleotide linkages are respectively between at least three consecutive positions in the 5' or 3' proximal terminal region of the second strand, whereby the proximal terminal region preferably adjoins the terminal region, and the positions of the one or more abasic nucleosides of the second strand are at least defined according to claim 11.

32. The inhibitor or inhibitor used according to claim 30 or 21, wherein the one or more phosphorothioate internucleotide linkages are respectively between at least three consecutive positions in the 5' and / or 3' terminal region of the first strand, whereby preferably the terminal positions of the 5' and / or 3' terminal region of the first strand are linked to their adjacent positions by phosphorothioate internucleotide linkages.

33. The inhibitor or inhibitor used according to any one of claims 10 to 32, 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 generally present in the terminal region of the second strand, preferably in its 3' terminal region.

34. The inhibitor or inhibitor used according to claim 33, wherein the ligand moiety comprises: i) one or more GalNAc ligands; and / or ii) one or more derivatives of GalNAc ligands; and / or iii) one or more GalNAc ligands and / or derivatives of GalNAc ligands conjugated to the SiRNA via a linker.

35. The inhibitor or inhibitor used according to claim 34, wherein the one or more GalNAc ligands and / or derivatives of GalNAc ligands are directly or indirectly conjugated to the 5' or 3' terminal region of the second strand of the siRNA oligomer, preferably in its 3' terminal region.

36. The inhibitor or inhibitor used according to claim 34 or 35, wherein the ligand moiety comprises:

37. The inhibitor or inhibitor used according to claim 34 or 35, having the structure: Wherein: R1 is independently selected from the group consisting of hydrogen, methyl and ethyl each time it appears; R2 is selected from the group consisting of: hydrogen, hydroxy, -OC 1-3 alkyl, -C(=O)OC 1-3 alkyl, halogen and nitro; Each occurrence of X1 and X2 is independently selected from the group consisting of methylene, oxygen, and sulfur; m is an integer from 1 to 6; n is an integer from 1 to 10; q, r, s, t, v are independently integers from 0 to 4, provided that: (i) both q and r cannot be 0 simultaneously; and (ii) s, t, and v cannot all be 0 simultaneously; Z is an oligomer.

38. The inhibitor or inhibitor used according to claim 34 or 35 has the structure: Wherein: r and s are independently integers selected from 1 to 16; and Z is an oligomer.

39. The inhibitor or inhibitor used according to one or more of the preceding claims, which is formulated with an excipient and / or a carrier into a pharmaceutical composition.

40. A pharmaceutical composition comprising the inhibitor according to one or more of the preceding claims, and a pharmaceutically acceptable excipient or carrier.

41. A pharmaceutical composition for treating diabetes, comprising the inhibitor according to one or more of the preceding claims, and a pharmaceutically acceptable excipient or carrier.

42. Use of B4GALT as a target in identifying one or more therapeutic agents for treating diabetes.

43. A method for treating or preventing diabetes, which comprises administering to a patient an inhibitor of post-translational glycosylation such as a B4GALT1 inhibitor, for example, the inhibitor defined according to one or more of the preceding claims.

44. B4GALT1 for use as a biomarker for diabetes.

45. Use of B4GALT1 in an in vivo method for predicting diabetes susceptibility, typically by monitoring the sequence and / or expression level and / or function of B4GALT1 in a sample obtained from a patient.

46. 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 a diagnosed patient.

47. Use of the inhibitor or composition according to one or more of the preceding claims in the preparation of a drug for treating diabetes.

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