Expression and / or function inhibitors

Abstract

The present application relates to inhibitors, compositions containing inhibitors, and uses of the inhibitors and compositions in the treatment or prevention of diseases associated with blood coagulation disorders, such as hemophilia.

Description

Technical Field

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

[0002] Inhibitors, such as oligonucleoside / oligonucleotide compounds, are inhibitors of gene expression and / or other target (e.g., lncRNA) expression or function, and 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 organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleosides / oligonucleotides that prevent protein formation through gene silencing.

[0003] Over the past two decades, a number of modified siRNA compounds have been developed for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutics for the treatment of a variety of diseases, including central nervous system diseases, inflammatory diseases, metabolic disorders, oncology, infectious diseases, and ocular diseases.

[0004] The present application relates to inhibitors, such as oligomers, such as nucleic acids, such as oligonucleoside / oligonucleotide compounds, and their use in treating and / or preventing diseases. Summary of the invention

[0005] The present application is defined in the claims and relates, inter alia:

[0006] In one aspect, the present application relates to inhibitors of ZPI expression and / or function, wherein the inhibitor is coupled to one or more ligand moieties.

[0007] In another aspect, the present application relates to the inhibitor according to the present application, wherein the inhibitor is a siRNA oligomer.

[0008] In another aspect, the present application relates to an inhibitor of ZPI expression and / or function, wherein the inhibitor is a siRNA oligomer.

[0009] In another aspect, the present application relates to an inhibitor according to the present application, wherein the inhibitor comprises a siRNA oligomer coupled to one or more ligand moieties.

[0010] In another aspect, the present application relates to use of the inhibitor according to the present application in preventing or treating diseases associated with coagulation disorders, such as hemophilia.

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

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

[0013] In another aspect, the present application relates to an inhibitor for use according to the present application, wherein the target of the inhibitor is ZPI.

[0014] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which is a siRNA oligomer having a first strand and a second strand, wherein:

[0015] i) the length of the first strand of the siRNA is in the range of 15-30 nucleosides, preferably 19-25 nucleosides, more preferably 23 or 25 nucleosides; even more preferably 23 nucleosides; and / or

[0016] ii) The length of the second strand of the siRNA is in the range of 15-30 nucleosides, preferably 19-25 nucleosides, more preferably 21 nucleosides.

[0017] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the second sense strand further comprises one or more abasic nucleosides in the terminal region of the second strand, and wherein the abasic nucleosides are linked to adjacent nucleosides via reverse internucleoside bonds.

[0018] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the second chain comprises:

[0019] i) the second strand terminal region has 2 or more abasic nucleosides; and / or

[0020] ii) the 5' or 3' terminal region of the second strand has 2 or more abasic nucleosides; and / or

[0021] iii) the 5' or 3' terminal region of the second strand has 2 or more abasic nucleosides, wherein the abasic nucleosides are present in a pendant form, for example as described herein; and / or

[0022] iv) the second strand terminal region has 2 or more consecutive abasic nucleosides, wherein preferably one such abasic nucleoside is the terminal nucleoside; and / or

[0023] v) there are 2 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

[0024] vi) at least one abasic nucleoside is linked to an adjacent abasic nucleoside via an inverted internucleoside linkage in the terminal region of the second strand; and / or

[0025] vii) at least one abasic nucleoside is linked to an adjacent abasic nucleoside via an inverted internucleoside linkage in the 5' or 3' terminal region of the second strand; and / or

[0026] viii) the penultimate nucleoside is an abasic nucleoside that is linked to the non-terminal nucleoside (referred to herein as the penultimate nucleoside) via an inverted bond; and / or

[0027] ix) an abasic nucleoside which acts as the two terminal nucleosides connected via a 5'-3' bond when the chain is read in the direction towards the end;

[0028] x) an abasic nucleoside which, when the chain is read in the terminal direction including the terminal nucleoside, serves as the two terminal nucleosides connected via a 3'-5' bond;

[0029] xi) an abasic nucleoside as the terminal 2 positions, wherein the penultimate nucleoside is linked to the penultimate nucleoside via the reverse bond, and wherein the reverse bond is a 5-5' reverse bond or a 3'-3' reverse bond;

[0030] xii) an abasic nucleoside as the terminal 2 positions, wherein the penultimate nucleoside is linked to the penultimate nucleoside via said reverse bond, and wherein

[0031] (1) the reverse bond is a 5-5' reverse bond, and when reading toward the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and penultimate abasic nucleoside is 3'5'; or

[0032] (2) The reverse bond is a 3-3' reverse bond, and when reading toward the end including the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 5'3'.

[0033] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the reverse internucleoside bond is located in the terminal region distal to the 5' region of the second chain, or in the terminal region distal to the 3' region of the second chain.

[0034] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the inverted internucleoside bond is a 3'3 inverted bond.

[0035] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the reversed internucleoside bond is a 5'5 reversed bond.

[0036] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein one or more nucleosides on the first strand and / or the second strand are modified to form modified nucleosides.

[0037] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the modification is a modification on the 2'-OH group of the ribose sugar, optionally selected from a 2'-Me or 2'-F modification.

[0038] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein counting from position 1 of the first strand, the first strand comprises 2'-F at position 14, position 2, position 6 or any combination thereof.

[0039] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, 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.

[0040] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the first strand and the second strand each comprise a 2'-Me and a 2'-F modification.

[0041] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which is a siRNA, wherein the siRNA comprises at least one thermally unstable modification, suitably at one or more positions of positions 1-9 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 1-9 of the first strand, wherein the unstable modification is selected from modified unlocked nucleic acids (UNA) and glycol nucleic acids (GNA), preferably glycol nucleic acids.

[0042] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the siRNA comprises at least one thermolabile modification at position 7 of the first strand, counting from position 1 of the first strand.

[0043] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which is a siRNA, wherein, counting from position 1 of the second strand, the siRNA comprises 3 or more 2'-F modifications at positions 7-13 of the second strand, such as 4, 5, 6 or 7 2'-F modifications at positions 7-13 of the second strand.

[0044] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which is a siRNA, wherein, counting from position 1 of the second strand, the second strand comprises at least 3, such as 4, 5 or 6 2'-Me modifications at positions 1-6 of the second strand.

[0045] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which is siRNA, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3'-terminal region, preferably including the terminal nucleoside of the 3'-terminal region, or at least within 1 or 2 nucleosides from the terminal nucleoside in the 3'-terminal region.

[0046] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which is siRNA, wherein the first strand comprises 7 consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside of the 3' terminal region.

[0047] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the siRNA oligomer further comprises one or more phosphorothioate internucleoside bonds.

[0048] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the one or more phosphorothioate internucleoside bonds are located between at least three consecutive positions of the 5' or 3' proximal terminal region of the second chain, respectively, whereby the proximal terminal region is preferably adjacent to the terminal region, wherein the one or more abasic nucleosides of the second chain are located in the terminal region.

[0049] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the one or more thiophosphate nucleoside bonds are respectively located between at least three consecutive positions in the 5' and / or 3' terminal region of the first chain, wherein preferably the terminal position of the 5' and / or 3' terminal region of the first chain is connected to its adjacent position by a thiophosphate nucleoside bond.

[0050] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the oligomer is a siRNA, and the second strand of the siRNA is directly or indirectly coupled 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.

[0051] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the ligand part comprises

[0052] i) one or more GalNAc ligands; and / or

[0053] ii) one or more GalNAc ligand derivatives; and / or

[0054] iii) one or more GalNAc ligands and / or GalNAc ligand derivatives coupled to the siRNA via a linker.

[0055] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly coupled to the 5' or 3' terminal region of the second strand of the siRNA oligomer, preferably to its 3' terminal region.

[0056] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, wherein the ligand part comprises

[0057]

[0058] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which has the following structure:

[0059]

[0060] in:

[0061] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[0062] R2 is selected from the group consisting of hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0063] X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;

[0064] m is an integer from 1 to 6;

[0065] n is an integer from 1 to 10;

[0066] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0067] (i) q and r cannot be zero at the same time; and

[0068] (ii) s, t and v cannot all be 0 at the same time;

[0069] Z is an oligomer.

[0070] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which has the following structure:

[0071]

[0072] in:

[0073] r and s are independently integers selected from 1-16; and

[0074] Z is an oligomer.

[0075] In another aspect, the present application relates to an inhibitor or an inhibitor for use according to the present application, which is formulated into a pharmaceutical composition with an excipient and / or a carrier.

[0076] In another aspect, the present application relates to a pharmaceutical composition comprising an inhibitor according to one or more of the aforementioned aspects, and a pharmaceutically acceptable excipient or carrier.

[0077] In another aspect, the present application relates to a pharmaceutical composition comprising the inhibitor according to the present application and a pharmaceutically acceptable excipient or carrier combination, and use thereof in treating diseases associated with coagulation disorders such as hemophilia.

[0078] In another aspect, the present application relates to targeting ZPI to identify one or more therapeutic agents for treating diseases associated with coagulation disorders, such as hemophilia.

[0079] In another aspect, the present application relates to a method for treating or preventing a disease associated with coagulation disorders, such as hemophilia, comprising administering to a patient a ZPI inhibitor, such as an inhibitor defined according to one or more of the preceding aspects.

[0080] In another aspect, the present application relates to the use of ZPI as a biomarker for diseases associated with coagulation disorders, such as hemophilia.

[0081] In another aspect, the present application relates to ZPIs for in vivo prediction of susceptibility to coagulation disorder-related diseases such as hemophilia, typically by monitoring the sequence and / or expression level and / or function of the ZPI in a sample obtained from a patient.

[0082] In another aspect, the present application relates to a method for predicting susceptibility to a disease associated with a coagulation disorder, such as hemophilia, and optionally a method for treating a disease associated with a coagulation disorder, such as hemophilia, in a patient, the method comprising:

[0083] (a) obtaining a sample from a patient,

[0084] (b) detecting the sequence and / or expression and / or function of the ZPI in said sample obtained from the patient,

[0085] (c) predicting susceptibility to a disease associated with a coagulation disorder, such as hemophilia, based on the sequence and / or expression and / or function of the ZPI in said sample obtained from the patient,

[0086] (d) preferably administering an effective amount of a ZPI inhibitor to the diagnosed patient.

[0087] In another aspect, the present application relates to use of the inhibitor or composition according to the present application in the preparation of a medicament for treating diseases associated with coagulation disorders, such as hemophilia. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1a Exemplary linear configurations of conjugates are shown.

[0089] Figure 1b Exemplary branching configurations of conjugates are shown.

[0090] Figure 2-5 Preferred oligomer-linker-ligand constructs of the present application are shown.

[0091] Figure 6 Details of the formula described in statements 1-101 disclosed herein are shown.

[0092] Figure 7 Details of the formulas described in clauses 1-56 disclosed herein are shown.

[0093] Figure 8 shows a typical pathway diagram of the coagulation cascade on the left (A), which is too simplistic to truly represent the biology. On the right (B) is an example of a network model that captures the full complexity of coagulation.

[0094] Fig. 9An overview of the network biology workflow is shown (top). (middle) Coagulation directed network. Directed network built using the ETX proprietary interactome, which includes directed interactions from NLP and curated pathway data. (bottom) Identification of targets on the coagulation network using KPA. On the right is a bar chart showing the top ranked proteins using the KPA approach. While the clinically validated hemophilia target AT3 is among the top 10 proteins, ZPI ranks higher based on the selected network metric. The heatmap panel on the left shows whether the protein is expressed and / or secreted in hepatocytes.

[0095] FIG. 10 shows the changes in the expression of hemophilia A in three different treatment groups (wild-type control group, hemophilia A mice receiving vehicle (0.9% saline), and hemophilia A mice receiving the GalNAc-siRNA construct ETXM1184) 3 days after injury ( Fig. 10A ) and 10 days ( Fig. 10B ) Visual bleeding scores. Definitions of bleeding scores are provided below.

[0096] Figure 11 shows (A) comparison of knee joint diameters on days 3 and 10 after injury in mice from three different treatment groups (wild-type control group, hemophilia A mice receiving vehicle (0.9% saline), and hemophilia A mice receiving the GalNAc-siRNA construct ETXM1184), and (B) comparison of skinned knee joint diameters on day 10 after injury in mice from the same three treatment groups.

[0097] FIG12 shows a comparison of the severity of the following in mice in three different treatment groups (wild-type control group, hemophilia A mice receiving vehicle (0.9% saline), and hemophilia A mice receiving the GalNAc-siRNA construct ETXM1184): (A) bone marrow hyperplasia, (B) osteoarthritis, (C) chondrocyte degeneration / necrosis, (D) hemorrhage, (E) hemosiderin deposition, (F) hematoma, (G) osteoclastic bone resorption, (H) osteolysis, (I) periostitis, (J) subchondral bone sclerosis, (K) tendinosis, (L) tendinitis, and (M) tenosynovitis ( Figure 12M ). DETAILED DESCRIPTION

[0098] The present application provides, inter alia, inhibitors, such as oligomers, such as nucleic acids, such as inhibitory RNA molecules (which may be referred to as iRNA or siRNA), and compositions containing them, which can affect the expression of a target, such as by binding to mRNA transcribed from a gene. The target can be in a cell, such as a cell in a subject (e.g., a human). The inhibitor can be used to prevent and / or treat diseases associated with, for example, target gene expression.

[0099] In particular, the present application identifies that ZPI inhibitors may be useful for preventing and / or treating diseases associated with coagulation disorders such as hemophilia.

[0100] Protein Z-dependent protease inhibitor (ZPI) is a protein that circulates in the blood and inhibits factors Xa and XIa of the coagulation cascade. It is a member of the class of serine protease inhibitors (serpins). In humans, ZPI is encoded by the ZPI gene (SEQ ID NO: 1).

[0101] SEQ ID NO:1(ZPI)

[0102]

[0103] The inventors employed network analysis, which enabled them to assign multiple genes or proteins to fewer driver processes; and to mine these processes for influential drug targets. The approach exploits information that is typically overlooked in standard gene set analysis - known and predicted interactions between genes (and proteins), and other genes contained in the same or related pathways. In particular, the inventors analyzed the human coagulation process using a network model that highlighted ZPI as a preferred target for hemophilia and highlighted an existing drug target - antithrombin 3 (AT3, the target of Fitusiran), thereby also validating the effectiveness of the network approach. Experimental confirmation that inhibiting ZPI is indeed a viable strategy for treating hemophilia is provided in Example 9.

[0104] The inhibition disclosed herein may be directed to a gene or a protein produced by expression of the gene, and reference to a ZPI specifically encompasses inhibition of gene expression or function, as well as inhibition of the protein product alone.

[0105] definition

[0106] "First strand" is also referred to herein as the antisense strand or guide strand, which can be used interchangeably herein and refers to a nucleic acid strand, such as a strand of an siRNA, such as a dsiRNA, which includes a region that is substantially complementary to a target sequence (e.g., mRNA). For example, as used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence). When the complementary region is not completely complementary to the target sequence, mismatches may occur in the interior or terminal regions of the molecule. In some embodiments, a double-stranded nucleic acid, such as an siRNA agent of the present application, includes nucleotide mismatches in the antisense strand.

[0107] "Second strand" (also referred to herein as the sense strand or passenger strand, and used interchangeably herein) refers to a nucleic acid strand, such as a siRNA, that includes a region that is substantially complementary to a region of the antisense strand as defined herein.

[0108] In the context of a molecule comprising a nucleic acid with a ligand moiety, optionally also with a linker moiety, the nucleic acid of the present application may be referred to as an oligonucleotide moiety or an oligonucleoside moiety.

[0109] Oligonucleotide is a short nucleic acid polymer. Although oligonucleotide contains phosphodiester bond between its nucleoside components (base sugaring), the application is not limited to the oligonucleotides always connected by this phosphodiester between adjacent nucleosides, and other nucleoside oligomers connected by non-phosphate bonds are also considered. For example, the bond between nucleotides can be a thiophosphate bond. Therefore, the term "oligonucleoside" herein covers oligonucleotides and other nucleoside oligomers. Oligonucleoside is a nucleic acid having at least a portion preferably an oligonucleotide according to the application. According to the application, it is also preferred to have one or more or most of phosphodiester backbone bonds between nucleosides. According to the application, it is also preferred to have one or more or most of phosphodiester backbone bonds between nucleosides, and it is also preferred to have one or more thiophosphate backbone bonds (usually in the terminal region of the first and / or second chain) between nucleosides.

[0110] In some embodiments, the double-stranded nucleic acid (eg, siRNA) agent of the present application includes a nucleoside mismatch in the sense strand. In some embodiments, the nucleoside mismatch is, for example, within 5, 4, 3, 2, or 1 nucleosides of the 3' end of the nucleic acid (eg, siRNA).

[0111] In another embodiment, the nucleoside is mismatched, for example, in the 3'-terminal nucleoside of the nucleic acid (eg, siRNA).

[0112] "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 primary transcription RNA processing, or may be a continuous portion of the nucleotide sequence of any RNA molecule (e.g., LNCRNA) that it is desired to inhibit.

[0113] The length of the target sequence can be about 10-35 nucleosides, such as about 15-30 nucleosides. For example, the target sequence can be about 15-30 nucleosides, 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 nucleosides in length. Ranges and lengths between the above listed ranges and lengths are also considered part of this application.

[0114] The term "ribonucleoside" or "nucleoside" may also refer to modified nucleosides, as described in detail below.

[0115] The nucleic acid may be DNA or RNA, and may contain modified nucleosides. RNA is the preferred nucleic acid.

[0116] The terms "iRNA", "siRNA", "RNAi agent", and "iRNA agent", "RNA interfering agent" are used interchangeably herein and refer to an agent containing RNA that mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. siRNA directs sequence-specific degradation of mRNA through RNA interference (RNAi).

[0117] Double-stranded RNA is referred to herein as "double-stranded siRNA (dsiRNA) agent", "double-stranded siRNAs (dsiRNAs) molecules", "double-stranded RNA (dsRNA) agent", "double-stranded RNA (dsRNA) molecule", "dsiRNA agent", "dsiRNA molecule" or "dsiRNA', which refers to a complex of ribonucleic acid molecules, having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as "sense" and "antisense" orientations relative to the target RNA. The majority of nucleosides in each strand of a nucleic acid, such as a dsRNA molecule, are preferably ribonucleosides, but in this case, each or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides or modified ribonucleosides. In addition, for example, as used in this specification, "siRNA" may include chemically modified ribonucleosides.

[0118] The term "modified nucleoside" refers to a nucleoside having an independently modified sugar moiety, a modified internucleoside linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleoside includes substitutions, additions, or removals of, for example, functional groups or atoms of an internucleoside linkage, a sugar moiety, or a nucleobase. For purposes of this specification and claims, any such modifications used in siRNA-type molecules are encompassed by "iRNA" or "RNAi agent" or "siRNA" or "siRNAi agent".

[0119] The double-stranded region of the nucleic acid of the present application, such as dsRNA, can range in length from about 9 to 40 base pairs, such as from 9 to 36 base pairs in length, such as from about 15 to 30 base pairs in length, such as from about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 base pairs in length, such as from 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, 15-19, 15-21, 15-22, 15-23 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.

[0120] The two strands that form the double-stranded structure can be different parts of a larger molecule or they can be separate molecules, such as RNA molecules.

[0121] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the double-stranded structure of a double-stranded nucleic acid. A ds nucleic acid may comprise an overhang of at least one nucleoside; alternatively, the overhang may comprise at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides or more. The nucleoside overhang may comprise or consist of nucleoside analogs, including deoxynucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the overhang / nucleoside may be present at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand.

[0122] In some embodiments, the antisense strand has 1-10 nucleosides, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside overhangs at the 3' end or the 5' end.

[0123] "Flat" or "blunt end" means that there is no unpaired nucleoside at the end of the double-stranded nucleic acid, that is, no nucleoside overhang. The nucleic acid of the present application includes nucleic acids without nucleoside overhang at one end or at both ends.

[0124] Unless otherwise indicated, the term "complementary" refers to the ability of the oligonucleosides comprising the first nucleoside sequence to hybridize with the oligonucleosides or polynucleosides comprising the second nucleoside sequence and to form a double-stranded structure under certain conditions, as understood by those skilled in the art, when used to describe the first nucleoside sequence related to the second nucleoside sequence. Such conditions can be, for example, stringent conditions, wherein stringent conditions can include: 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50 ℃ or 70 ℃ for 12-16 hours, then washing (see, for example, Molecular Cloning:A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press).

[0125] For example, as described herein, the complementary sequence in a nucleic acid (e.g., dsiRNA) comprises a base pairing of an oligonucleoside or polynucleoside of a first nucleoside sequence and an oligonucleoside or polynucleoside comprising a second nucleoside sequence over the entire length of one or two nucleoside sequences. Such sequences may be referred to herein as "completely complementary" to each other. However, when the first sequence is referred to herein as "substantially complementary" or "partially complementary" to the second sequence, the two sequences may be completely 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 final application, such as inhibiting gene expression via the RISC pathway. In determining complementarity, overhangs should not be considered mismatches. For example, a nucleic acid, such as a dsRNA, comprises an oligonucleotide of 17 nucleosides in length and an oligonucleoside of 19 nucleosides of another length, wherein the longer oligonucleoside comprises 17 nucleoside sequences that are completely complementary to the shorter oligonucleoside, and may still be referred to as "completely complementary".

[0126] For example, as used herein, a "complementary" sequence may also include or be completely formed by non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleosides, as long as the above-mentioned requirements for its hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0127] The terms "complementary," "fully complementary," and "substantially / partially complementary" herein may apply 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.

[0128] In the present application, the second strand of the nucleic acid according to the present application, in particular the dsiRNA for inhibiting ZPI, is at least partially complementary to the first strand of the nucleic acid. In some embodiments, the first and second strands of the nucleic acid described herein are partially complementary if they form a double-stranded region of at least 17 base pairs in length and containing no more than 1, 2, 3, 4 or 5 mismatched base pairs.

[0129] In some embodiments, the first and second strands of the nucleic acids described herein are partially complementary if they form a double-stranded region of 19 base pairs in length and contain no more than 1, 2, 3, 4, or 5 mismatched base pairs. In some embodiments, the first and second strands of the nucleic acids described herein are partially complementary if they form a double-stranded region of 21 base pairs in length and contain no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0130] Alternatively, the first and second strands of the nucleic acid described herein are partially complementary if they form a double-stranded region of at least 17 base pairs in length, wherein at least 14, 15, 16 or 17 of the base pairs are complementary base pairs, in particular Watson Crick base pairs.

[0131] In some embodiments, if the first and second strands of the nucleic acid described herein form a double-stranded region of 19 base pairs in length, they are partially complementary, wherein at least 14, 15, 16, 17, 18 or all 19 base pairs are complementary base pairs, in particular Watson Crick base pairs. In some embodiments, if the first and second strands of the nucleic acid described herein form a double-stranded region of 21 base pairs in length, they are partially complementary, wherein at least 16, 17, 18, 19, 20 or all 21 base pairs are complementary base pairs, in particular Watson Crick base pairs. For example, 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 polynucleotide that is substantially or partially complementary to a continuous portion of a target mRNA (e.g., an mRNA encoding a gene). In some embodiments, the continuous portion of the mRNA is a sequence listed in Table 1, i.e., any one of SEQ ID NOs: 2-121. For example, a polynucleotide is complementary to at least a portion of a target gene mRNA if the sequence is substantially or partially complementary to a non-interrupted portion of an mRNA encoding a target gene.

[0132] Therefore, in some preferred embodiments, the antisense oligonucleotides disclosed herein are completely complementary to the target gene sequence.

[0133] In other embodiments, the antisense oligonucleosides disclosed herein are substantially or partially complementary to a target RNA sequence and comprise a contiguous nucleoside sequence that is at least about 80% complementary to an equivalent region of a 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.

[0134] In some embodiments, the first (antisense) strand of a nucleic acid according to the present application is partially or fully complementary to a consecutive portion of RNA transcribed from a ZPI gene. In some embodiments, the first strand of a nucleic acid according to the present application is partially or fully complementary to a consecutive portion of at least 17 nucleosides of a ZPI mRNA. In some embodiments, the first strand of a nucleic acid according to the present application is partially or fully complementary to a consecutive portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of a ZPI mRNA. In some embodiments, the first strand of a nucleic acid according to the present application is partially or fully complementary to a consecutive portion of 17, 18, 19, 20, 21, 22 or 23 nucleosides of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-121.

[0135] In some embodiments, if the first (antisense) strand of a nucleic acid according to the present application comprises a contiguous nucleoside sequence of at least 17 nucleosides, and wherein at least 14, 15, 16 or 17 nucleosides of the contiguous nucleoside sequence are complementary to a contiguous portion of a ZPI mRNA, it is partially complementary to a contiguous portion of a ZPI mRNA. In some embodiments, the first strand of a nucleic acid according to the present application comprises a contiguous nucleoside sequence of at least 17 nucleosides, wherein at least 14, 15, 16 or 17 nucleosides of the contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-121. In some embodiments, the first strand of a nucleic acid according to the present application comprises a contiguous nucleoside sequence of 19 nucleosides, wherein at least 14, 15, 16, 17, 18 or all 19 nucleosides of the contiguous nucleoside sequence are complementary to a contiguous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-121. In some embodiments, the first strand of the nucleic acid according to the present application comprises a continuous nucleoside sequence of 23 nucleosides, wherein at least 18, 19, 20, 21, 22 or all 23 nucleosides of the continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2-121.

[0136] In some embodiments, the nucleic acids described herein, such as siRNAs, comprise a sense strand that is substantially or partially complementary to an antisense polynucleoside, which in turn is complementary to a target gene sequence and comprises a contiguous nucleoside sequence that is at least about 80% complementary to an equivalent region of the nucleoside sequence of the antisense strand over its entire length, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0137] In some embodiments, the nucleic acid of the present application, such as siRNA, comprises an antisense strand that is substantially or partially complementary to a target sequence, and comprises a contiguous nucleotide sequence that is at least 80% complementary to the target sequence over its entire length, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary, or 100% complementary.

[0138] As used herein, when the target gene sequence has sufficient complementarity with a nucleic acid (e.g., iRNA) agent to promote target knockdown, "subject" refers to an animal, such as a mammal, including primates (e.g., humans, non-human primates, such as monkeys and chimpanzees), or non-primates or birds that endogenously or heterologously express the target gene. In some preferred embodiments, the subject is a human.

[0139] The term "treating" or "treatment" refers to a beneficial or desired result, including but not limited to alleviating or ameliorating one or more symptoms associated with gene expression. "Treatment" may also mean prolonging survival compared to expected survival without treatment. Treatment may include preventing the development of complications, such as reducing liver damage in subjects with liver infection.

[0140] As used herein, a "therapeutically effective amount" is intended to include an amount of a nucleic acid (e.g., an iRNA) that, when administered to a patient for treating a subject suffering from a disease, is sufficient to affect the treatment of the disease (e.g., by reducing, ameliorating, or maintaining the existing disease or one or more symptoms of the disease or its associated complications).

[0141] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, pursuant to a reasonable benefit / risk ratio.

[0142] As used herein, the term "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 of the formulation and not injurious to the subject being treated.

[0143] When a value or range of values ​​is mentioned for a parameter, the values ​​and ranges between the stated values ​​are also intended to be part of the present application.

[0144] As used herein, the articles "a" and "an" refer to one or more than one (ie, to at least one) of the grammatical object of the article.

[0145] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0146] Unless the context clearly indicates otherwise, the term "or" is used herein to mean and can be used interchangeably with the term "and / or." For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand or sense strand and antisense strand."

[0147] As used herein, the term "about" means within the typical tolerance range of the art. For example, "about" can be understood as about 2 standard deviations from the mean. In some embodiments, about means +10%. In some embodiments, about means +5%. When about appears before a series of numbers or ranges, it is understood that "about" can modify each number in the series or range.

[0148] For example, the context clearly indicates that the term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least", as well as all subsequent numbers or integers that can be logically included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides in a nucleic acid molecule of 21 nucleosides" means that 18, 19, 20 or 21 nucleosides have the specified properties. When at least appears before a series of numbers or ranges, it is understood that "at least" can modify each number in the series or range.

[0149] For example, as used herein, "not more than" or "less than" refers to the value adjacent to the phrase and the value or integer logically lower to zero, depending on the logic of the context. For example, a double strand with "not more than 2 nucleosides" overhang has 2, 1 or 0 nucleosides overhang. When "not more than" appears before a series of numbers or ranges, it can be understood that "not more than" can modify each number in the series or range.

[0150] The terminal region of a strand is the last 5 nucleotides at the 5' or 3' end.

[0151] The nucleobase sequence is the sequence of nucleic acid bases in an oligomer.

[0152] Those skilled in the art can appropriately combine various embodiments of the present application.

[0153] Target

[0154] The inhibition target disclosed herein may be, but is not limited to, mRNA, polypeptide, protein or gene.

[0155] These are targets whose inhibition could be useful in preventing or treating diseases associated with coagulation disorders such as hemophilia.

[0156] The target of inhibition is ZPI, and inhibition can be achieved by inhibiting the expression or function of ZPI gene or protein or both.

[0157] In a preferred embodiment, the target site is located in the mRNA expressed by the ZPI gene. Exemplary target sequences on the ZPI mRNA are listed in Table 1 below.

[0158] Table 1

[0159]

[0160]

[0161]

[0162] It will be appreciated that SEQ ID NOs: 2-121 relate to human (human) mRNA sequences.

[0163] Disease / condition

[0164] The present application relates to an inhibitor suitable for or used in treating diseases associated with coagulation disorders such as hemophilia.

[0165] Hemophilia is a primarily inherited genetic disorder that impairs the body's ability to form blood clots, a process needed to stop bleeding. This causes subjects to bleed longer after an injury, bruise easily, and have an increased risk of bleeding into the joints or brain. Patients with milder forms of the disease may only experience symptoms after an accident or during surgery. Bleeding into the joints can lead to permanent damage, while bleeding into the brain can cause long-term headaches, seizures, or decreased levels of consciousness.

[0166] There are two main types of hemophilia: hemophilia A, which occurs due to low levels of clotting factor VIII, and hemophilia B, which occurs due to low levels of clotting factor IX. They are usually inherited from a parent through the X chromosome, which carries a non-functional gene. Rarely, hemophilia may develop later in life due to new mutations that may arise during early development, or due to the formation of antibodies against clotting factors. Other types include hemophilia C, which occurs due to low levels of factor XI, von Willebrand disease, which occurs due to low levels of a substance called von Willebrand factor, and parahemophilia, which occurs due to low levels of factor V. Hemophilia A, B, and C prevent the intrinsic coagulation pathway from functioning properly; this clotting pathway is necessary when the lining of the blood vessels is damaged. Acquired hemophilia is associated with cancer, autoimmune diseases, and pregnancy. Diagnosis is made by testing the blood's ability to clot and the levels of clotting factors.

[0167] In some embodiments, the inhibitors described herein are suitable for treating or are used to treat hemophilia A, B, and / or C. In some embodiments, the inhibitors described herein are suitable for treating or are used to treat hemophilia A and / or B. In some embodiments, the inhibitors described herein are suitable for treating or are used to treat acquired hemophilia. In some embodiments, the inhibitors described herein are suitable for treating or are used to treat von Willebrand disease. In some embodiments, the inhibitors described herein are suitable for treating or are used to treat parahemophilia.

[0168] Without wishing to be bound by theory, for example as shown in Figure 10, treatment with the inhibitors described herein can increase the level of coagulation factors, thereby reducing or preventing bleeding. Therefore, in a preferred embodiment, treatment with the inhibitors described herein can reduce or prevent bleeding episodes in subjects with hemophilia. In another preferred embodiment, treatment with the inhibitors described herein can reduce or prevent bleeding in the joints of hemophiliacs. In some embodiments, treatment with the inhibitors described herein can reduce or prevent bleeding in the muscles or brain of hemophiliacs.

[0169] Alternatively or additionally, treatment of a subject, preferably a subject suffering from a coagulation disorder such as hemophilia, with an inhibitor as described herein may result in one or more of the following:

[0170] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce bone marrow proliferation. Fig. 12A As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced myeloproliferation in said mice.

[0171] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce osteoarthritis. Fig. 12B As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced osteoarthritis in the mice.

[0172] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce chondrocyte degeneration / necrosis. Fig. 12C As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced chondrocyte degeneration / necrosis in the mice.

[0173] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce major bleeding. Fig.12D As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced major bleeding in the mice.

[0174] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce hemosiderin deposition. Fig.12E As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced hemosiderin deposition in the mice.

[0175] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce the occurrence of hematomas. Fig.12F As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced hematomas in the mice.

[0176] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce osteoclastic bone resorption. Figure 12G As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced osteoclastic bone resorption in the mice.

[0177] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce osteolysis. Fig.12H As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced osteolysis in the mice.

[0178] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce periostitis. Fig.12IAs shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced periostitis in the mice.

[0179] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce subchondral bone sclerosis. Fig.12J As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced subchondral bone sclerosis in the mice.

[0180] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce tendinosis. Figure 12K As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced tendinosis in said mice.

[0181] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce tendinitis. Figure 12L As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced tendinitis in the mice.

[0182] In some embodiments, treatment of a subject, preferably a subject with a coagulation disorder such as hemophilia, with an inhibitor as described herein can reduce tenosynovitis. Figure 12M As shown, treatment of hemophilia A mice with the inhibitor of the present application significantly reduced tenosynovitis in the mice.

[0183] Thus, in a particular embodiment, the present application relates to an inhibitor suitable for use in the treatment of, or for use in the treatment of, hemophilia, wherein the treatment of hemophilia is characterized by reduced bleeding and one or more of: reduced bone marrow proliferation, reduced osteoarthritis, reduced chondrocyte degeneration / necrosis, reduced bleeding, reduced hemosiderin deposition, reduced hematomas, reduced osteoclastic bone resorption, reduced osteolysis, reduced periostitis, reduced subchondral bone sclerosis, reduced tendon degeneration, reduced tendinitis and / or reduced tenosynovitis.

[0184] For example, as used herein, the term "treatment" refers to the treatment of any human or other vertebrate subject in need of a drug. The subject is expected to have undergone a physical examination by a medical practitioner or a veterinary practitioner, who has given a preliminary or definitive diagnosis indicating that the use of the specific treatment is beneficial for treating the disease of the human or other vertebrate. The timing and purpose of the treatment may vary from individual to individual, depending on the health status of the subject. Thus, the treatment may be preventive, palliative, symptomatic and / or curative.

[0185] Inhibitors

[0186] The inhibitors of the present application include nucleic acids (such as siRNA), antibodies and antigen-binding fragments thereof (such as monoclonal antibodies), polypeptides, antibody-drug conjugates and small molecules. Preferred are nucleic acids, such as siRNA.

[0187] Certain preferred features of the inhibitors of the present invention are given below, where the inhibitors are oligonucleotides, such as siRNA.

[0188] In some embodiments, the nucleic acid comprises a first strand comprising a sequence at least partially complementary to a portion of RNA transcribed from a ZPI gene (SEQ ID NO: 1). In a preferred embodiment, the nucleic acid comprises a first strand comprising a sequence at least partially complementary to a ZPI mRNA (NM_016186.3).

[0189] In some embodiments, the nucleic acid for inhibiting ZPI expression comprises a double-stranded region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand is:

[0190] (i) is at least partially complementary to a portion of the RNA transcribed from the ZPI gene, and

[0191] (ii) comprises at least 17 consecutive nucleosides that differ from any one of SEQ ID NOs: 122-241 by 0 or 1 nucleoside.

[0192] In some embodiments, the first strand comprises nucleosides 2-18 of any one of the sequences listed in SEQ ID NOs: 122-241.

[0193] In some embodiments, the first strand comprises any one of SEQ ID NOs: 122-241.

[0194] In some embodiments, the nucleic acid for inhibiting ZPI expression comprises a double-stranded region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand is:

[0195] (i) is at least partially complementary to a portion of the RNA transcribed from the ZPI gene, and

[0196] (ii) comprises at least 21 consecutive nucleotides that differ from any one of SEQ ID NOs: 122-241 by 0 or 1 nucleotide.

[0197] In some embodiments, the first strand comprises nucleosides 2-22 of any one of the sequences listed in SEQ ID NOs: 122-241.

[0198] In some embodiments, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs from any one of SEQ ID NOs: 242-361 by 0 or 1 nucleoside; wherein the second strand has a region that is at least 85% complementary to the first strand in the 17 consecutive nucleosides.

[0199] In some embodiments, the second strand comprises a nucleoside sequence of at least 19 consecutive nucleosides that differs from any one of SEQ ID NOs: 242-361 by 0 or 1 nucleoside; wherein the second strand has a region of at least 85% complementarity with the first strand over the 19 consecutive nucleosides.

[0200] In some embodiments, the second strand comprises a nucleoside sequence of at least 21 consecutive nucleosides that differs from any one of SEQ ID NOs: 242-361 by 0 or 1 nucleoside; wherein the second strand has a region of at least 85% complementarity with the first strand over the 21 consecutive nucleosides.

[0201] In some embodiments, the second strand comprises any one of SEQ ID NOs: 242-361.

[0202] In some embodiments, the nucleic acid comprises a first strand comprising, consisting of, or consisting essentially of a nucleotide sequence that differs from any one of SEQ ID NOs: 122-241 by 0 or 1 nucleotide;

[0203] and a second strand comprising, consisting of, or consisting essentially of a nucleotide sequence that differs from any one of SEQ ID NOs: 242-361 by 0 or 1 nucleotide.

[0204] It is preferred herein that the double-stranded region is formed between the first (antisense) strand and the complementary second (sense) strand. Table 2 below lists exemplary pairings of complementary antisense strands and sense strands:

[0205] Table 2

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] In a specific embodiment, the present application relates to a nucleic acid comprising a first and a second strand, the first and the second strand comprising, consisting of or consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0212] Unmodified first strand Unmodified second strand SEQ ID NO:148 SEQ ID NO:268 SEQ ID NO:145 SEQ ID NO:265 SEQ ID NO:144 SEQ ID NO:264 SEQ ID NO:165 SEQ ID NO:285 SEQ ID NO:202 SEQ ID NO:322

[0213] In some embodiments, the nucleic acid for inhibiting ZPI expression comprises a double-stranded region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand is:

[0214] (i) is at least partially complementary to a portion of the RNA transcribed from the ZPI gene, and

[0215] (ii) comprises at least 17 consecutive nucleosides that differ from any one of SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797 by 0 or 1 nucleoside.

[0216] In some embodiments, the first strand comprises nucleosides 2-18 of any of the sequences listed in SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797.

[0217] In some embodiments, the nucleic acid for inhibiting ZPI expression comprises a double-stranded region comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand is:

[0218] (i) is at least partially complementary to a portion of the RNA transcribed from the ZPI gene, and

[0219] (ii) comprises at least 21 consecutive nucleosides that differ from any one of SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797 by 0 or 1 nucleoside.

[0220] In some embodiments, the first strand comprises nucleosides 2-22 of any of the sequences listed in SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797.

[0221] In some embodiments, the first strand comprises any one of SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797.

[0222] Table 3 below summarizes the modification patterns of the nucleic acids set forth in SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797:

[0223] Table 3

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] In some embodiments, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs from any one of SEQ ID NOs: 562-761, 772-781, or 798-800 by 0 or 1 nucleoside; wherein the second strand has a region that is at least 85% complementary to the first strand at the 17 consecutive nucleosides.

[0233] In some embodiments, the second strand comprises a nucleoside sequence of at least 19 consecutive nucleosides that differs from any one of SEQ ID NOs: 562-761, 772-781, or 798-800 by 0 or 1 nucleoside; wherein the second strand has a region of at least 85% complementarity with the first strand over the 19 consecutive nucleosides.

[0234] In some embodiments, the second strand comprises a nucleoside sequence of at least 21 consecutive nucleosides that differs from any one of SEQ ID NOs: 562-761, 772-781, or 798-800 by 0 or 1 nucleoside; wherein the second strand has a region of at least 85% complementarity with the first strand over the 21 consecutive nucleosides.

[0235] In some embodiments, the second strand comprises any one of SEQ ID NOs: 562-761, 772-781, or 798-800.

[0236] Table 4 below summarizes the modification patterns of the nucleic acids set forth in SEQ ID NOs: 562-761, 772-781, or 798-800:

[0237] Table 4

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] For example, as used herein, particularly in Tables 3 and 4, the following abbreviations are used for modified nucleosides:

[0247] Am represents 2'-O-methyl-adenosine, Cm represents 2'-O-methyl-cytidine, Gm represents 2'-O-methyl-guanosine, Um represents 2'-O-methyl-uridine, Af represents 2'-fluoro-adenosine, Cf represents 2'-fluoro-cytidine, Gf represents 2'-fluoro-guanosine and Uf represents 2'-fluoro-uridine.

[0248] 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-methyl-adenosine nucleosides, which are connected by a 3'5' phosphorothioate bond. Abbreviations are not used to represent nucleosides connected by standard 3'5' phosphodiester bonds. For example, the abbreviation "AmAm" is used for two consecutive 2'-O-methyl-adenosine nucleosides, which are connected by a 3'5' phosphodiester bond.

[0249] In some embodiments, the nucleic acid comprises a first strand comprising, consisting of, or consisting essentially of a (modified) nucleoside sequence that differs by 0 or 1 nucleoside from any one of SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797;

[0250] and a second strand comprising, consisting of or consisting essentially of a (modified) nucleoside sequence that differs by 0 or 1 nucleoside from any one of SEQ ID NOs: 562-761, 772-781, or 798-800.

[0251] Preferred combinations of complementary modified antisense (first) and sense (second) strands are listed in Table 5 below:

[0252] Table 5

[0253]

[0254]

[0255]

[0256]

[0257]

[0258] In a particularly preferred embodiment, the present application 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 nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0259] Double chain ID Modified first strand Modified second strand ETXM342 SEQ ID NO:388 SEQ ID NO:588 ETXM339 SEQ ID NO:385 SEQ ID NO:585 ETXM338 SEQ ID NO:384 SEQ ID NO:584 ETXM359 SEQ ID NO:405 SEQ ID NO:605 ETXM396 SEQ ID NO:442 SEQ ID NO:642

[0260] In a more preferred embodiment, the present application 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 nucleotide sequence that differs by 0 or 1 nucleotide from any of the following first and second sequences:

[0261] Double chain ID Modified first strand Modified second strand Duplex ID Modified first strand Modified second strand ETXM1180 SEQ ID NO:762 SEQ ID NO:772 ETXM1181 SEQ ID NO:763 SEQ ID NO:773 ETXM1184 SEQ ID NO:764 SEQ ID NO:774 ETXM1185 SEQ ID NO:765 SEQ ID NO:775 ETXM1157 SEQ ID NO:766 SEQ ID NO:776 ETXM1162 SEQ ID NO:767 SEQ ID NO:777 ETXM1188 SEQ ID NO:768 SEQ ID NO:778 ETXM1189 SEQ ID NO:769 SEQ ID NO:779 ETXM1192 SEQ ID NO:770 SEQ ID NO:780 ETXM1193 SEQ ID NO:771 SEQ ID NO:781 ETXM1194 SEQ ID NO:782 SEQ ID NO:773 ETXM1195 SEQ ID NO:783 SEQ ID NO:775 ETXM1196 SEQ ID NO:784 SEQ ID NO:777 ETXM1197 SEQ ID NO:785 SEQ ID NO:779 ETXM1198 SEQ ID NO:786 SEQ ID NO:781

[0262] If there is an ambiguity between the sequence in this specification and the sequence in the attached sequence listing, the sequence provided in this specification is deemed to be the correct sequence.

[0263] Abasic nucleotides

[0264] In some embodiments, there is 1, such as 2, such as 3, such as 4 or more abasic nucleosides in the nucleic acids according to the present application. Abasic nucleosides are modified nucleosides because they lack the bases usually seen at position 1 of the sugar moiety. Typically, there will be a hydrogen at position 1 of the sugar moiety of the abasic nucleosides present in the nucleic acids according to the present application.

[0265] The abasic nucleoside is located in the terminal region of the second strand, preferably within the terminal 5 nucleosides of the strand end. The terminal region may be the terminal 5 nucleosides, which include the abasic nucleoside.

[0266] As preferred features, the second chain may include (all such combinations are specifically contemplated unless mutually exclusive):

[0267] The terminal region of the second strand has 2 or more abasic nucleosides; and / or

[0268] The 5' or 3' terminal region of the second strand has 2 or more abasic nucleosides; and / or

[0269] The second strand has 2 or more abasic nucleosides in the 5' or 3' terminal region, wherein the abasic nucleosides are present in a pendant form as described herein; and / or

[0270] The terminal region of the second strand has 2 or more consecutive abasic nucleosides, wherein preferably one such abasic nucleoside is the terminal nucleoside; and / or

[0271] There are 2 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

[0272] In the terminal region of the second strand, at least one abasic nucleoside is linked to an adjacent abasic nucleoside via an inverted internucleoside bond; and / or

[0273] In the 5' or 3' terminal region of the second strand, at least one abasic nucleoside is linked to an adjacent abasic nucleoside via an inverted internucleoside bond; and / or

[0274] At the penultimate nucleoside position is an abasic nucleoside linked via an inverted bond to a nucleoside other than the terminal nucleoside (referred to herein as the penultimate nucleoside); and / or

[0275] an abasic nucleoside which, when the chain is read in the terminal direction including the terminal nucleoside, serves as two terminal nucleosides joined via a 5'-3' bond;

[0276] an abasic nucleoside which, when the chain is read in the terminal direction including the terminal nucleoside, serves as two terminal nucleosides joined via a 3'-5' bond;

[0277] at the terminal 2 positions is an abasic nucleoside, wherein the penultimate nucleoside is linked to the penultimate nucleoside via the reverse bond, and wherein the reverse bond is a 5-5' reverse bond or a 3'-3' reverse bond;

[0278] At the terminal 2 positions are abasic nucleosides, wherein the penultimate nucleoside is linked to the penultimate nucleoside via the reverse bond, and wherein either

[0279] (1) the reverse bond is a 5-5' reverse bond, and when reading toward the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and penultimate abasic nucleoside is 3'5'; or

[0280] (2) The reverse bond is a 3-3' reverse bond, and when reading toward the end including the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 5'3'.

[0281] Preferably, there is an abasic nucleoside at the terminus of the second strand.

[0282] In the terminal region of the second strand, preferably at the terminal and penultimate position, there are preferably 2 or at least 2 abasic nucleosides.

[0283] Preferably, 2 or more abasic nucleosides are consecutive, for example, all abasic nucleosides may be consecutive. For example, the terminal 1 or terminal 2 or terminal 3 or terminal 4 nucleotides may be abasic nucleosides.

[0284] Abasic nucleosides may also be linked to adjacent nucleosides via 5'-3' phosphodiester bonds or reverse linkages, unless there is only 1 abasic nucleoside at the end, in which case it will be reverse-linked to the adjacent nucleoside.

[0285] Reverse linkages (also referred to as reverse linkages or reverse connections, as also found in the art) include 5'-5', 3-'3', 3'-2' or 2'-3' phosphodiester bonds between adjacent sugar moieties of nucleosides.

[0286] Non-terminal abasic nucleosides will have 2 phosphodiester bonds, one to each adjacent nucleoside, and these bonds may be inverted, 5'-3' phosphodiester bonds, or one of each.

[0287] Preferred embodiments include comprising two abasic nucleosides at the terminal and penultimate positions of the second strand, and wherein the reversed internucleoside bond is located between the penultimate (abasic) nucleoside and the penultimate nucleoside.

[0288] Preferably, there are 2 abasic nucleosides at the terminal and penultimate positions of the second strand, the penultimate nucleoside being linked to the penultimate nucleoside by an inverted internucleoside bond and to the terminal nucleoside by a 5'-3' or 3'-5' phosphodiester bond (reading in the terminal direction of the molecule).

[0289] The different preferred features are as follows:

[0290] The reverse internucleoside bond is a 3'-3' reverse bond. The reverse internucleoside bond is located in the terminal region away from the 5'-terminal phosphate group of the second chain.

[0291] The reverse internucleoside bond is a 5'-5' reverse bond. The reverse internucleoside bond is located in the terminal region away from the 3' terminal hydroxyl group of the second chain.

[0292] In some embodiments, the second strand comprises 2 consecutive abasic nucleosides in the 5'-terminal region of the second strand, wherein one such abasic nucleoside is the terminal nucleoside of the 5'-terminal region of the second strand and the other abasic nucleoside is the penultimate nucleoside of the 5'-terminal region of the second strand, wherein: (a) the penultimate abasic nucleoside is linked to the adjacent first basic nucleoside in the adjacent 5' proximal region by a reverse internucleoside bond; and (b) the reverse bond is a 5-5' reverse bond; and (c) when reading toward the end comprising the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 3'5'. More typically, (i) the first strand and the second strand each have a length of 23 nucleosides; (ii) there are two phosphorothioate internucleoside bonds between three consecutive positions in the 5' proximal region of the second strand, wherein in the region near the 5' end of the second strand, there is a first phosphorothioate internucleoside bond between the adjacent first basic nucleoside and the adjacent second basic nucleoside, and in the region near the 5' end of the second strand, there is a second phosphorothioate internucleoside bond between the adjacent second basic nucleoside and the adjacent third basic nucleoside. (iii) there are two phosphorothioate internucleoside bonds between three consecutive positions of the 5' and 3' terminal regions of the first chain, such that the terminal nucleosides of the 5' and 3' terminal regions of the first chain are respectively linked to the second-to-last nucleosides adjacent to the respective 5' and 3' positions through phosphorothioate internucleoside bonds, and the first second-to-last nucleoside of each 5' and 3' position is linked to the third-to-last nucleoside adjacent to the corresponding 5' and 3' position through phosphorothioate internucleoside bonds; and (iv) the second chain of the nucleic acid is directly or indirectly coupled to one or more ligand moieties at its 3' terminal region.

[0293] Alternatively, the second strand comprises two consecutive abasic nucleosides, preferably pendant in the 3' region of the second strand, wherein one of such abasic nucleosides is the terminal nucleoside of the 3' region of the second strand and the other abasic nucleoside is the penultimate nucleoside of the 3' region of the second strand, wherein (a) the penultimate abasic nucleoside is linked to the adjacent first basic nucleoside in the adjacent 3' proximal region by a reverse internucleoside bond; and (b) the reverse bond is a 3-3' reverse bond; and (c) when reading towards the end comprising the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate basic nucleoside is 5'-3'. More typically, (i) the first strand and the second strand each have a length of 23 nucleosides; (ii) there are two phosphorothioate internucleoside bonds between three consecutive positions in the region near the 3' end of the second strand, wherein a first phosphorothioate internucleoside bond exists between an adjacent first basic nucleoside and an adjacent second basic nucleoside in the region near the 3' end of the second strand, and a second phosphorothioate internucleoside bond exists between an adjacent second basic nucleoside and an adjacent third basic nucleoside in the region near the 3' end of the second strand; iii) there are two phosphorothioate internucleoside bonds between three consecutive positions of the 5' and 3' terminal regions of the first chain, such that the terminal nucleosides of each of the 5' and 3' terminal regions of the first chain are respectively linked to the second-to-last nucleosides adjacent to the respective 5' and 3' positions through phosphorothioate internucleoside bonds, and the first second-to-last nucleosides of each 5' and 3' position are linked to the third-to-last nucleosides adjacent to the respective 5' and 3' positions through phosphorothioate internucleoside bonds; and (iv) the second chain of the nucleic acid is directly or indirectly coupled to one or more ligand moieties at its 5' terminal region.

[0294] Examples of structures are as follows (the specific RNA nucleosides shown are not limiting and can be any RNA nucleosides):

[0295] A 3'-3' reverse bond (also shows the 5'-3 orientation of the last phosphodiester bond between two abasic molecules read towards the ends of the molecule)

[0296]

[0297] B illustrates the 5'-5' reverse bond (and shows the 3'-5' orientation of the last phosphodiester bond between two abasic molecules read toward the ends of the molecule)

[0298]

[0299] One or more abasic nucleosides present in nucleic acid provide in the presence of one or more reverse internucleoside bonds, i.e. 5'-5' or 3'-3' reverse internucleoside bonds. Reverse connection occurs due to the orientation change of adjacent nucleoside sugars, so that sugar radical has a 3'-5' direction, rather than traditional 5'-3' direction (with reference to the numbering of nucleoside sugar ring atoms). One or more abasic nucleosides present in nucleic acid of the application preferably include this reverse nucleoside sugar.

[0300] If the terminal nucleoside has reverse orientation, this will result in " reverse " terminal configuration of whole nucleic acid. Although some structures drawn and quoted herein have used conventional 5'-3' direction (with reference to the numbering of nucleoside sugar ring atoms), it is understandable that there is a terminal nucleoside with a change of direction and a near-end 3'-3' reverse bond, which will result in nucleic acid having an overall 5'-5' end structure (i.e. conventional 3' terminal nucleoside becomes 5' terminal nucleoside). Or, it is understandable that there is a terminal nucleoside with a change of direction and a near-end 5'-5' reverse bond, which will result in nucleic acid having an overall 3'-3' end structure.

[0301] Near-end 3'-3' or 5'-5' reverse bonds as described herein can include the reverse bonds directly adjacent to / connected to the terminal nucleosides with reverse orientation, such as single terminal nucleosides with reverse orientation.Or, near-end 3'-3' or 5'-5' reverse bonds as described herein can include the reverse bonds adjacent to 2 or more nucleosides with reverse orientation, such as 2 or more terminal region nucleosides with reverse orientation, such as terminal and penultimate nucleosides.In this way, the reverse bonds can be connected to the penultimate nucleosides with reverse orientation.Although it will be appreciated by those skilled in the art that reverse orientation as described above can cause nucleic acid molecules to have overall 3'-3' or 5'-5' end structures such as described herein, it should also be appreciated that in the presence of one or more additional reverse connections and / or nucleosides with reverse orientation, the whole nucleic acid may have the 3'-5' end structures corresponding to the 5' / 3' ends of conventional positioning.

[0302] In one aspect, the nucleic acid can have a 3'-3' inverted linkage and the terminal sugar moiety can comprise a 5' OH rather than a 5' phosphate group at the 5' position of the terminal sugar.

[0303] Thus, it will be clearly understood by those skilled in the art that 5'-5', 3'-3' and 3'-5' (reading along this terminal direction) terminal variants of the more conventional 5'-3' structures (numbered with reference to the ring atoms on the terminal nucleoside sugar) described herein are included within the scope of the present application wherein one or more inverted bonds are present.

[0304] For example, in the reverse internucleoside bond and / or one or more nucleosides with reverse orientation, reverse ends are produced, and when the relative position of connection (for example with joint) or the position of internal features (for example modified nucleosides) is defined with respect to the 5' or 3' end of nucleic acid, then the 5' or 3' end refers to the conventional 5' or 3' end when there is no reverse connection, and wherein the conventional 5' or 3' end is determined by considering the directionality and / or nucleoside orientation of most internal nucleoside bonds in nucleic acid. It can be judged from the direction of these internal bonds and / or nucleosides that, in the absence of reverse connection, which ends of nucleic acid will constitute the conventional 5' and 3' ends of molecule (with reference to the ring atom numbering on the terminal nucleoside sugar).

[0305] For example, in the structure shown below, there are abasic residues in the first 2 positions at the "5'" end. When the orientation of the terminal nucleosides is reversed, the "5'" end shown in the figure below, i.e., the conventional 5' end, may actually contain a 3'OH given the inversion of the nucleosides in the terminal position. However, when reading in the standard 5'[PO4] to 3'[OH] orientation of nucleic acid molecules (with reference to the numbering of the ring atoms on the nucleoside sugars), most molecules will contain a conventional internucleoside bond from the 3'OH of the sugar to the 5' phosphate of the next sugar, which can be used to determine the conventional 5' and 3' ends that would be found in the absence of the reversed terminal configuration.

[0306] A5'AA-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me 3'

[0307] The reverse bond is preferably located at the end of the nucleic acid (eg RNA), which is distal to the ligand portion of the molecule, eg the GalNAc-containing portion.

[0308] GalNAc-siRNA constructs having 5'-GalNAc on the sense strand may have an inverted linkage at the other end of the sense strand.

[0309] GalNAc-siRNA constructs having 3'-GalNAc on the sense strand may have an inverted linkage at the other end of the sense strand.

[0310] Nucleic acid length

[0311] In one aspect, i) the length of the first strand of the nucleic acid is in the range of 17-30 nucleosides, preferably 19-25 nucleosides, more preferably 19 or 23 nucleosides; and / or ii) the length of the second strand of the nucleic acid is in the range of 17-30 nucleosides, preferably 19-25 nucleosides, more preferably 19 or 21 nucleosides.

[0312] Typically, the double-stranded region of the nucleic acid is between 17 and 30 nucleotides in length, more preferably 19 or 21 nucleotides in length. Similarly, the complementary region between the first strand and the RNA portion transcribed from the ZPI gene is between 17 and 30 nucleotides in length. Typically, the double-stranded structure of a nucleic acid (e.g., iRNA) is about 15-30 base pairs in length, 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, 19-3 21-24, 21-23 or 21-22 base pairs in length. Ranges and lengths between the above ranges and lengths are also considered to be part of this application.

[0313] Similarly, the length of the complementary region between the antisense sequence and the target sequence and / or the complementary region between the antisense sequence and its sense sequence is about 15-30 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 21-22, 21-23, or 21-22 nucleosides in length. Ranges and lengths between the above ranges and lengths are also considered to be part of this application.

[0314] In some preferred embodiments, the complementary region between the antisense sequence and the target sequence and / or the complementary region between the antisense sequence and its sense sequence is at least 17 nucleosides in length. For example, the complementary region between the antisense strand and the target is 19-21 nucleosides in length, for example, the complementary region is 21 nucleosides in length.

[0315] In a preferred embodiment, each strand is no more than 30 nucleosides in length.

[0316] In some preferred embodiments, the double-stranded structure of the nucleic acid, such as siRNA, is 19 or 21 base pairs in length. In a particularly preferred embodiment, the double-stranded structure may have one of the following structures:

[0317] For example, ETXM316–ETXM415, ETXM436–ETXM515, and ETXM1064-ETXM1198:

[0318]

[0319] For example, ETXM416–ETXM435:

[0320]

[0321] Nucleic acids as described herein, such as dsRNA, can also include one or more single-stranded nucleoside overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3 or 4 nucleosides. The nucleoside overhang can include or consist of nucleosides / nucleoside analogs, including deoxynucleosides / nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. In addition, the overhanging nucleosides can be present at the 5' end, 3' end or both ends of the antisense or sense strand of the nucleic acid (e.g., dsRNA).

[0322] In some preferred embodiments, at least one strand comprises a 3' overhang of at least 1 nucleoside, for example, 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.

[0323] Nucleic acid modification

[0324] In some embodiments, the nucleic acids described herein, such as RNA, such as dsiRNA, do not comprise further modifications, such as chemical modifications or conjugations known in the art and described herein.

[0325] In other preferred embodiments, the nucleic acids described herein, such as RNA, such as dsiRNA, are further chemically modified to enhance stability or other beneficial properties.

[0326] In some embodiments of the present application, substantially all of the nucleosides are modified.

[0327] The nucleic acids in the present application can be synthesized or modified by methods well known in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference.

[0328] Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation, coupling, reverse linkage) or 3'-terminal modifications (coupling, DNA nucleosides within RNA or RNA nucleosides within DNA, reverse linkage, etc.); base modifications, such as substitution with stable bases, unstable bases, or bases that can pair with more partners, coupling bases; sugar modifications (e.g., at the 2'-position or 4'-position) or replacement of sugar groups; or backbone modifications, including modification or replacement of phosphodiester bonds.

[0329] Specific nucleic acid examples, such as siRNA compounds useful in embodiments described herein, include but are not limited to RNAs containing modified backbones or without natural internucleoside bonds. Nucleic acids with modified backbones, such as RNA, include those without phosphorus atoms in the backbone. For the purposes of this specification, and as sometimes cited in the art, modified nucleic acids, such as RNAs without phosphorus atoms in their internucleoside backbones, may also be considered oligonucleotides. In some embodiments, modified nucleic acids (e.g., siRNAs) have phosphorus atoms in their internucleoside backbones.

[0330] Modified nucleic acids, such as RNA backbones, include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters and borate esters with normal 3'-5' bonds, 2'-5' linked analogs, and those with opposite polarity, wherein adjacent nucleoside unit pairs are linked 5'-3' or 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0331] Modified nucleic acids, such as RNA, can also contain one or more substituted sugar moieties. Nucleic acids described herein, such as siRNAs, such as dsiRNAs, can include one of the following at the 2'-position: OH; F, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted. 2'-O-methyl and 2'-F are preferred modifications.

[0332] In some preferred embodiments, the nucleic acid comprises at least one modified nucleoside.

[0333] The nucleic acid of the present application may comprise one or more modified nucleosides on the first strand and / or the second strand.

[0334] In some embodiments, substantially all nucleosides of the sense strand and all nucleosides of the antisense strand comprise modifications.

[0335] In some embodiments, all nucleosides of the sense strand and substantially all nucleosides of the antisense strand comprise modifications.

[0336] In some embodiments, all nucleosides of the sense strand and all nucleosides of the antisense strand comprise a modification.

[0337] 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, wherein Me is methoxy), 2'-fluoro modified nucleosides, 2'-deoxy-modified nucleosides, locked nucleosides, unlocked nucleosides, conformationally constrained nucleosides, constrained ethyl nucleosides, abasic nucleosides, 2'-amino-modified nucleosides, 2'-O-allyl modified nucleosides, 2'-C-alkyl-modified nucleosides, 2'-hydroxyl-modified nucleosides, 2'-methoxyethyl-modified nucleosides, 2'-O-alkyl-modified nucleosides, morpholino nucleosides, phosphoramidates, non-natural bases comprising nucleosides, tetrahydropyran modified nucleosides, 1,5-anhydrohexitol modified nucleosides, cyclohexenyl modified nucleosides, nucleosides comprising thiophosphate groups, nucleosides comprising methylphosphonate groups, nucleosides comprising 5'-phosphates, and nucleosides comprising 5'-phosphate mimetics. In another embodiment, the modified nucleosides comprise a short sequence of 3'-terminal deoxythymidine (dT).

[0338] The modification of nucleosides may preferably be selected from the group consisting of, but not limited to, LNA, HNA, CeNA, 2-methoxyethyl, 2'-O-alkyl, 2-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In another embodiment, the modification on the nucleosides is a 2-O-methyl ("2-Me") or 2'-fluoro modification.

[0339] A preferred modification is modification of the 2'-OH group of the ribose sugar moiety, optionally selected from 2'-Me or 2'-F modifications.

[0340] Preferred nucleic acids comprise one or more nucleosides on the first strand and / or the second strand which are modified to form modified nucleosides as follows:

[0341] A nucleic acid, wherein the modification is a modification on the 2'-OH group of the ribose sugar moiety, optionally selected from a 2'-Me or 2'-F modification.

[0342] A nucleic acid, wherein the first strand comprises a 2'-F modification at position 2, position 6, position 14, or any combination thereof, counting from position 1 of the first strand,

[0343] A nucleic acid, wherein the second strand comprises a 2'-F modification at position 7, position 9, position 11, or any combination thereof, counting from position 1 of the second strand.

[0344] A nucleic acid, wherein the second strand comprises a 2'-F modification at position 7 and / or 9, and / or 11, and / or 13, counted from position 1 of the second strand.

[0345] A nucleic acid wherein the second strand comprises a 2'-F modification at positions 7, 9 and 11, counted from position 1 of the second strand.

[0346] A nucleic acid, wherein the first strand and the second strand each comprise a 2'-Me and a 2'-F modification.

[0347] A nucleic acid comprising at least one thermally labile modification, suitably located at one or more positions in positions 1-9 of the first strand, counting from position 1 of the first strand, and / or located at one or more positions on the second strand aligned with positions 1-9 of the first strand, wherein the labile modification is selected from modified unlocked nucleic acids (UNA) and glycol nucleic acids (GNA), preferably glycol nucleic acids. A nucleic acid, wherein, counting from position 1 of the second strand, the nucleic acid comprises 3 or more 2'-F modifications at positions 7-13 of the second strand, for example, 4, 5, 6 or 7 2'-F modifications at positions 7-13 of the second strand.

[0348] A nucleic acid wherein, counting from position 1 of the second strand, the second strand comprises at least 3, such as 4, 5 or 6, 2'-Me modifications at positions 1-6 of the second strand.

[0349] A nucleic acid, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside of the 3' terminal region, or at least within 1 or 2 nucleosides from the terminal nucleoside in the 3' terminal region.

[0350] A nucleic acid, wherein the first strand comprises seven consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside of the 3' terminal region.

[0351] A nucleic acid, counting from position 1 of a first strand, comprising at least one thermolabile modification at position 7 of the first strand.

[0352] A nucleic acid which is a siRNA oligonucleotide, counting from position 1 of the second strand, wherein the siRNA oligonucleotide comprises at least 3 2'-F modifications at positions 6-12 of the second strand.

[0353] A nucleic acid which is a siRNA oligonucleotide, counting from position 1 of the second strand, wherein the second strand comprises at least 3 2'-Me modifications at positions 1-6 of the second strand.

[0354] A nucleic acid which is a siRNA oligonucleoside, wherein each of the first and second strands comprises an alternating modification pattern, preferably a completely alternating modification pattern along the entire length of each of the first and second strands, wherein the nucleosides of the first strand are modified to: (i) 2'Me modifications on odd-numbered nucleosides counting from position 1 of the first strand, and (ii) 2'F modifications on even-numbered nucleosides counting from position 1 of the first strand, and the nucleosides of the second strand are modified to: (i) 2'F modifications on odd-numbered nucleosides counting from position 1 of the second strand, and (ii) 2'Me modifications on even-numbered nucleosides counting from position 1 of the second strand. Typically, this completely alternating modification pattern is present in a blunt-ended oligonucleotide wherein each of the first and second strands is 19 or 23 nucleosides in length.

[0355] Position 1 of the first or second strand is the nucleoside closest to the end of the nucleic acid (ignoring any abasic nucleosides) and is joined to the adjacent nucleoside (at position 2) by a 3'-5' internal bond, referenced to the bonds between the backbone sugar moieties, and read in the direction away from the end of the molecule.

[0356] Thus, it can be seen that "position 1 of the sense strand" is the nucleoside most 5' to the conventional 5' end of the sense strand (excluding abasic nucleosides). Typically, the nucleoside at position 1 of the sense strand will be equal to the 5' nucleoside of the selected target nucleic acid sequence, and more generally, starting from position 1 of the sense strand, the nucleosides of the sense strand will be identical to the nucleosides of the target nucleic acid sequence, while also allowing for acceptable mismatches between sequences.

[0357] For example, as used herein, "position 1 of the antisense strand" is the 5'most nucleoside at the conventional 5' end of the antisense strand (excluding abasic nucleosides). As described above, there will be a complementary region between the sense strand and the antisense strand, and thus the antisense strand will also have a region complementary to the target nucleic acid sequence described above.

[0358] In some embodiments, the nucleic acid, such as an RNAi agent, further comprises at least one phosphorothioate or methylphosphonate internucleoside bond. For example, the phosphorothioate or methylphosphonate internucleoside bond can be located at the 3'-end or the terminal region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands, the sense strand and the antisense strand.

[0359] In some embodiments, the phosphorothioate or methylphosphonate internucleoside linkage is located at the 5' end or terminal region of one strand (ie, the sense strand or the antisense strand); or at the termini of both strands, the sense strand and the antisense strand.

[0360] In some embodiments, the phosphorothioate or methylphosphonate internucleoside linkages are located at the 5' and 3'-ends, or at the terminal region of one strand (ie, the sense strand or the antisense strand); or at the ends of both strands, the sense strand and the antisense strand.

[0361] Any nucleic acid can comprise one or more phosphorothioate (PS) modifications within the nucleic acid, such as at least two PS internucleoside linkages at the chain termini.

[0362] At least one oligoribonucleoside chain preferably comprises at least two consecutive phosphorothioate modifications in its last three nucleosides.

[0363] Therefore, the present application also relates to: a nucleic acid disclosed herein, which comprises a phosphorothioate internucleoside bond between at least two or three consecutive positions, respectively, for example in the 5' and / or 3' terminal region and / or the proximal terminal region of the second chain, wherein the proximal terminal region is preferably adjacent to the terminal region, and in the second chain, the one or more abasic nucleosides are located in the terminal region.

[0364] The present application discloses a nucleic acid, which comprises phosphorothioate internucleoside bonds between at least two or three consecutive positions in the 5' and / or 3' end region of the first chain, wherein preferably, the terminal position of the 5' and / or 3' end region of the first chain is connected to its adjacent position by a phosphorothioate internucleoside bond.

[0365] The nucleic acid strand may be RNA comprising interthionucleoside bonds between three nucleosides adjacent to two terminal abasic nucleosides.

[0366] The preferred nucleic acid is a double-stranded RNA comprising two adjacent abasic nucleosides at the 5' end of the second strand and a ligand moiety of one or more GalNAc ligand moieties at the opposite 3' end of the second strand. More preferably, the same nucleic acid may also comprise a phosphorothioate bond between nucleotides at positions 3-4 and 4-5 of the second strand, reading from position 1 of the second strand. More preferably, the same nucleic acid may also comprise a 2'F modification at positions 7, 9 and 11 of the second strand.

[0367] Preferred modifications of nucleic acids having this structure are as follows:

[0368]

[0369] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any of the following:

[0370] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0371] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0372] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0373] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0374] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me.

[0375] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0376] Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0377] Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0378] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0379] Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0380] Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0381] Me–Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or

[0382] Me–Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0383] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0384] Me–Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or

[0385] Me–Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me,

[0386] where (s) is a phosphorothioate internucleoside bond.

[0387] A nucleic acid, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following:

[0388] ia-ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or

[0389] ia-ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0390] ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0391] ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0392] ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0393] Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, or

[0394] Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or

[0395] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or

[0396] Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or

[0397] Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia,

[0398] 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 exists in the form of a 2-nucleoside overhang.

[0399] A nucleic acid, wherein the modified nucleoside of the second strand comprises a modification pattern (5'-3') according to any one of the following:

[0400] 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

[0401] 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

[0402] 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

[0403] 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

[0404] ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or

[0405] Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia, or

[0406] Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or

[0407] Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or

[0408] Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or

[0409] Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia,

[0410] in:

[0411] (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 chain, the inverted abasic nucleoside is present in a pendant form of 2 nucleosides.

[0412] A nucleic acid wherein the modified nucleosides comprise any of the following modification patterns:

[0413] Modification mode 1: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, first strand (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me

[0414] Or modification mode 2: second link (5'-3'): Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first link (5'-3'): Me-F-Me-F-Me-F-Me-Me-FF-Me-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me

[0415] Or modification mode 3: second link (5'-3'): Me-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first link (5'-3'): Me-F-Me-F-Me-F-Me-Me-FF-Me-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me

[0416] Or modification mode 4: second link (5'-3'): Me-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first link (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me

[0417] Modification model 5: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me , 1st pin (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

[0418] Modification model 6: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First pin (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.

[0419] One kind of nucleic acid, which includes the modified nucleic acids, and the following modified model intermediate functions:

[0420] 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

[0421] 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

[0422] 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-M, 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

[0423] 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

[0424] 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

[0425] 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-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

[0426] Where (s) is a phosphorothioate internucleoside bond.

[0427] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0428] 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

[0429] 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

[0430] 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

[0431] 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

[0432] 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

[0433] 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

[0434] where (s) is a phosphorothioate internucleoside linkage.

[0435] A nucleic acid, wherein the modified nucleoside comprises any one of the following modification patterns:

[0436] 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

[0437] 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

[0438] Or modification mode 3: second link (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first link (5'-3'): Me-F-Me-F-Me-F-Me-Me-F-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me

[0439] Or modification mode 4: second link (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first link (5'-3'): Me-F-Me-F-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me

[0440] Or modification mode 5: second link (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first link (5'-3'): Me-F-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me

[0441] Or modification mode 6: second link (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first link (5'-3'): Me-F-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-Me-Me-Me-Me,

[0442] It shows that there is no opposite direction.

[0443] One kind of nucleic acid, which includes the modified nucleic acids, and the following modified model intermediate functions:

[0444] Modification mode 1: Second link (5'-3'): e-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, First link (5'-3'): Me-F-Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-Me-Me-Me

[0445] 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

[0446] 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

[0447] 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

[0448] 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

[0449] 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,

[0450] Wherein ia represents an inverted abasic nucleoside. When the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside exists in the form of a 2-nucleoside overhang.

[0451] One kind of nucleic acid, which includes the modified nucleic acids, and the following modified model intermediate functions:

[0452] Modification pattern 1: Second pin (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F-Me-Me , 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0453] Modification model 2: Second pin (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me , 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0454] Modification model 3: Second pin (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-M e, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0455] Modification model 4: Second pin (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me-Me , 1st pin (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

[0456] Modification model 5: Second pin (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-M e, 1st pin (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

[0457] Modification model 6: Second pin (5'-3'): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-M e, 1st pin (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

[0458] Among them:

[0459] (s)It is one kind of sulfur base, one kind of sulfuric acid core, one kind of ia representative without any direction.

[0460] One kind of nucleic acid, which includes the modified nucleic acids, and the following modified model intermediate functions:

[0461] Modification pattern 1: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-FFFFF-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia , 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0462] Modification pattern 2: Second pin (5'-3'): Me-Me-Me-Me-Me-FF-Me-FFFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia , 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0463] Modification model 3: Second pin (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFFF-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-i a, 1st pin (5'-3'): Me(s)F(s)Me-F-Me-F-Me-FF-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me

[0464] Or modification mode 4: second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFFF-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

[0465] Or modification mode 5: second strand (5'-3'): Me-Me-Me-Me-Me-Me-Me-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-i a, 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

[0466] Or modification pattern 6: second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-i a, 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

[0467] 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 nucleotide is present in a 2-nucleoside overhang form.

[0468] 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, provided that the total number of 2'F sugar modifications in the first strand does not consist of 4 or 6 2'F modifications.

[0469] 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, wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5 or 7 2'F modifications.

[0470] 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, wherein the total number of 2'F sugar modifications in the first strand consists of 3 2'F modifications.

[0471] 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, wherein the total number of 2'F sugar modifications in the first strand consists of 5 2'F modifications.

[0472] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0473] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0474] wherein X2, X3 and X4 are selected from 2'Me and 2'F sugar modifications, with the proviso 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.

[0475] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0476] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0477] wherein X2 is a 2'F sugar modification, and X3 and X4 are 2'Me sugar modifications.

[0478] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0479] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0480] wherein X3 is a 2'F sugar modification, and X2 and X4 are 2'Me sugar modifications.

[0481] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0482] Me–F–Me–X2–Me–F–(Me)7–(F–Me)2–X3–Me–X4–(Me)3

[0483] wherein X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications.

[0484] 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, wherein the total number of 2'F sugar modifications in the first strand consists of 7 2'F modifications.

[0485] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0486] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0487] wherein X2, X3 and X4 are selected from 2'Me and 2'F sugar modifications, provided that for X2, X3, X4, at least one is a 2'F sugar modification and the other two sugar modifications are 2'Me sugar modifications.

[0488] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0489] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0490] wherein X2 is a 2'F sugar modification, and X3 and X4 are 2'Me sugar modifications.

[0491] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0492] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0493] wherein X3 is a 2'F sugar modification, and X2 and X4 are 2'Me sugar modifications.

[0494] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0495] Me–F–Me–X2–Me–F–Me–(F)2–(Me)4–(F–Me)2–X3–Me–X4–(Me)3

[0496] wherein X4 is a 2'F sugar modification, and X2 and X3 are 2'Me sugar modifications.

[0497] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0498] Me–F–(Me)3–X1–(Me)7–F–Me–F–(Me)7

[0499] Among them, X1 is a thermally unstable modification.

[0500] A nucleic acid wherein the first strand comprises the following 2' sugar modification pattern (5'-3'):

[0501] Me–F–(Me)3–X1–Me–(F)2–(Me)4–F–Me–F–(Me)7

[0502] Among them, X1 is a thermally unstable modification.

[0503] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0504] (Me)8–(F)3–(Me) 10.

[0505] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0506] (Me)8–(F)3–(Me) 10 ,as well as

[0507] 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 4 or 6 2'F modifications.

[0508] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0509] (Me)8–(F)3–(Me) 10 ,as well as

[0510] 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 wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5 or 7 2'F modifications.

[0511] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene, and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0512] (Me)8–(F)3–(Me) 10 ,as well as

[0513] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0514] Me–F–(Me)3–X1–(Me)7–F–Me–F–(Me)7, where X1 is a thermally labile modification.

[0515] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0516] (Me)8–(F)3–(Me) 10 ,as well as

[0517] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0518] (Me–F)3–(Me)7–F–Me–F–(Me) 7.

[0519] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0520] (Me)8–(F)3–(Me) 10 ,as well as

[0521] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0522] Me–F–(Me)3–F–(Me)7–(F–Me)2–F–(Me) 5.

[0523] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0524] (Me)8–(F)3–(Me) 10 ,as well as

[0525] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0526] Me–F–(Me)3–F–(Me)7–F–Me–F–(Me)3–F–(Me)3.

[0527] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0528] (Me)8–(F)3–(Me) 10 ,as well as

[0529] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0530] Me–F–(Me)3–X1–Me–(F)2–(Me)4–F–Me–F–(Me)7, where X1 is a thermally labile modification.

[0531] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0532] (Me)8–(F)3–(Me) 10 ,as well as

[0533] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0534] (Me–F)3–Me–(F)2–(Me)4–(F–Me)2–(Me)6.

[0535] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0536] (Me)8–(F)3–(Me) 10 ,as well as

[0537] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0538] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–F–(Me)5.

[0539] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar modification pattern (5'-3'):

[0540] (Me)8–(F)3–(Me) 10 ,as well as

[0541] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0542] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–(Me)2–F–(Me)3.

[0543] A nucleic acid wherein the second strand comprises a 2' sugar and the following abasic modification pattern (5'-3'):

[0544] ia-ia-(Me)8–(F)3–(Me) 10

[0545] Where ia represents an inverted abasic nucleoside.

[0546] A nucleic acid wherein the second strand comprises a 2' sugar and the following abasic modification pattern (5'-3'):

[0547] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0548] 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 4 or 6 2'F modifications.

[0549] A nucleic acid wherein the second strand comprises a 2' sugar and the following abasic modification pattern (5'-3'):

[0550] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0551] 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, wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5 or 7 2'F modifications.

[0552] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0553] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0554] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0555] Me–F–(Me)3–X1–(Me)7–F–Me–F–(Me)7, where X1 is a thermally labile modification.

[0556] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0557] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0558] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0559] (Me–F)3–(Me)7–F–Me–F–(Me)7.

[0560] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0561] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0562] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0563] Me–F–(Me)3–F–(Me)7–(F–Me)2–F–(Me)5.

[0564] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0565] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0566] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0567] Me–F–(Me)3–F–(Me)7–F–Me–F–(Me)3–F–(Me)3.

[0568] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0569] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0570] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0571] Me–F–(Me)3–X1–Me–(F)2–(Me)4–F–Me–F–(Me)7,

[0572] Among them, X1 is a thermally unstable modification.

[0573] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0574] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0575] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0576] (Me–F)3–Me–(F)2–(Me)4–(F–Me)2–(Me)6.

[0577] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0578] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

[0579] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0580] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–F–(Me)5.

[0581] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0582] ia-ia-(Me)8–(F)3–(Me) 10 , wherein ia represents an inverted abasic nucleoside; and

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

[0584] Me–F–(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–(Me)2–F–(Me)3.

[0585] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0586] ia-ia-Me(s)Me(s)(Me)6–(F)3–(Me) 10 ,

[0587] wherein ia represents an inverted abasic nucleoside, and (s) represents a phosphorothioate bond.

[0588] A nucleic acid wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0589] 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

[0590] 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 4 or 6 2'F modifications.

[0591] A nucleic acid, wherein the second strand comprises the following 2' sugar modification pattern (5'-3'):

[0592] 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

[0593] 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, wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5 or 7 2'F modifications.

[0594] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0595] 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

[0596] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0597] Me(s)F(s)(Me)3–X1–(Me)7–F–Me–F–(Me)5(s)Me(s)Me, where X1 is a thermally labile modification.

[0598] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0599] 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

[0600] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0601] Me(s)F(s)Me–F–Me–F–(Me)7–F–Me–F–(Me)5(s)Me(s)Me.

[0602] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0603] 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

[0604] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0605] Me(s)F(s)(Me)3–F–(Me)7–(F–Me)2–F–(Me)3(s)Me(s)Me.

[0606] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0607] 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

[0608] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0609] Me(s)F(s)(Me)3–F–(Me)7–F–Me–F–(Me)3–F–Me(s)Me(s)Me.

[0610] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0611] 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

[0612] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0613] 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 thermally labile modification.

[0614] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0615] 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

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

[0617] Me(s)F(s)Me–F–Me–F–Me–(F)2–(Me)4–(F–Me)2–(Me)4(s)Me(s)Me.

[0618] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0619] 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

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

[0621] Me(s)F(s)(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–F–(Me)3(s)Me(s)Me.

[0622] A nucleic acid comprising a first strand at least partially complementary to a portion of an RNA transcribed from a target gene and a second strand at least partially complementary to the first strand, wherein the first and second strands form a double-stranded region of at least 17 nucleosides in length, and wherein the nucleosides of the second strand comprise the following 2' sugar and abasic modification pattern (5'-3'):

[0623] 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

[0624] The nucleosides of the first strand contain the following 2' sugar modification pattern (5'-3'):

[0625] Me(s)F(s)(Me)3–F–Me–(F)2–(Me)4–(F–Me)2–(Me)2–F–Me(s)Me(s)Me.

[0626] Preferred modifications are as follows:

[0627] Modification mode 1:

[0628] 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,

[0629] 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 thermally unstable modification.

[0630] Or modifier mode 2:

[0631] 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,

[0632] 1st pin (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;

[0633] Modification model 3:

[0634] Second pin (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,

[0635] 1st pin (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;

[0636] Modification model 4:

[0637] Second pin (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,

[0638] 1st pin (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;

[0639] Modification model 5:

[0640] Second pin (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,

[0641] 1st pin (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, in which X1 is heatless fixation;

[0642] Modification model 6:

[0643] Second pin (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,

[0644] 1st pin (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;

[0645] Modification model 7:

[0646] Second pin (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;

[0647] 1st pin (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;

[0648] Modification model 8:

[0649] Second pin (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,

[0650] First pin (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.

[0651] Special selection of training below:

[0652] Ceremony model 1:

[0653] Second pin (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,

[0654] First link (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, in which X1 is a hot-fixed modification;

[0655] A modification model 2:

[0656] Second pin (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,

[0657] 1st pin (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;

[0658] Modification model 3:

[0659] Second pin (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,

[0660] 1st pin (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;

[0661] Modification model 4:

[0662] Second pin (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,

[0663] 1st pin (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;

[0664] Modification model 5:

[0665] Second pin (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,

[0666] 1st pin (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, in which X1 is heat failure fixation;

[0667] Modification model 6:

[0668] Second pin (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,

[0669] 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

[0670] Or with modifier mode 7:

[0671] 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

[0672] 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;

[0673] Or in modifier mode 8:

[0674] 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,

[0675] 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;

[0676] wherein (s) is a phosphorothioate internucleoside linkage.

[0677] Conjugation of nucleic acids and ligands

[0678] Another modification of the nucleic acids (e.g., RNA, e.g., siRNA) of the present application involves linking the nucleic acid (e.g., siRNA) to one or more ligand moieties, e.g., to enhance the activity, cellular distribution or cellular uptake of the nucleic acid (e.g., siRNA), e.g., into cells.

[0679] In some embodiments, the ligand moiety can be attached to a nucleic acid (e.g., a siRNA oligonucleotide) 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, such as covalently connecting two parts of a compound.

[0680] The ligand can be attached to the 3' or 5' end of the sense strand.

[0681] The ligand is preferably coupled to the 3' end of the sense strand of a nucleic acid, such as a siRNA agent.

[0682] Therefore, the present application relates in another aspect to a conjugate for inhibiting the expression of a target (eg, a target gene) in a cell, the conjugate comprising a nucleic acid portion and one or more ligand portions, wherein the nucleic acid portion comprises the nucleic acid disclosed herein.

[0683] In one aspect, the second strand of the nucleic acid is coupled directly or indirectly (eg, via a linker) to one or more ligand moieties, wherein the ligand moiety is typically present at the terminal region of the second strand, preferably at its 3' terminal region.

[0684] In some embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative attached to a nucleic acid (eg, dsiRNA) via a linker.

[0685] Therefore, the present application relates to a conjugate, wherein the ligand part comprises

[0686] i) one or more GalNAc ligands; and / or

[0687] ii) one or more GalNAc ligand derivatives; and / or

[0688] iii) one or more GalNAc ligands coupled to the nucleic acid via a linker.

[0689] The GalNAc ligand may be coupled directly or indirectly to the 5' or 3' terminal region of the second nucleic acid strand, preferably to the 3' terminal region thereof.

[0690] GalNAc ligands are well known in the art and are described inter alia in EP 3 775 207 A1.

[0691] In some embodiments, the ligand moiety comprises one or more ligands.

[0692] In some embodiments, the ligand moiety comprises one or more carbohydrate ligands.

[0693] In some embodiments, the one or more carbohydrates can be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and / or a polysaccharide.

[0694] In some embodiments, 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.

[0695] In some embodiments, the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[0696] In some embodiments, the compounds described herein comprise two or three N-acetylgalactosamine moieties.

[0697] In some embodiments, the one or more ligands are attached in a linear configuration or a branched configuration, for example, each configuration is separately attached to a branch point in the overall linker.

[0698] Exemplary linear configurations and exemplary branched configurations are as follows Figure 1a and 1b As shown:

[0699] exist Figure 1a In the above, (linear), (a) and / or (b) may generally represent a linking bond or group, such as a phosphate or thiophosphate group.

[0700] exist Figure 1b In some embodiments, the one or more ligands are connected in a bifurcated or trifurcated branched structure. Generally, a trifurcated branched configuration is more popular, such as an N-acetylgalactosamine trifurcated branched configuration.

[0701] Connectors

[0702] Exemplary compounds of the present application include a "linker moiety", such as that shown in formula (I), which is part of the entire "linker".

[0703] Formula (I)

[0704]

[0705] in:

[0706] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[0707] R2 is selected from the group consisting of hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0708] X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;

[0709] m is an integer from 1 to 6;

[0710] n is an integer from 1 to 10;

[0711] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0712] (i) q and r cannot be 0 at the same time; and

[0713] (ii) s, t and v cannot be 0 at the same time;

[0714] Z is an oligonucleoside moiety.

[0715] For example, as will be further understood in the art, the exemplary compounds of the present application include an overall linker located between the oligonucleoside portion and the ligand portion of these compounds. The overall linker thus "connects" the oligonucleoside portion and the ligand portion to each other.

[0716] The total linker is usually theoretically conceived to include one or more linker building blocks. For example, there is a linker portion, described as a "linker portion", as shown in formula (I), located near the ligand portion, and the ligand portion is usually directly or indirectly connected to the oligonucleoside portion through a branch point. For example, the linker portion described by formula (I) is also commonly referred to as the "ligand arm" of the total linker. There may also be (but not always) another connecting portion between the oligonucleoside portion and the branch point, which is usually referred to as the "tethering portion" of the total linker, and the oligonucleoside portion is "tethered" to the rest of the coupling compound. This "ligand arm" and / or "linker portion" and / or "tethering portion" can be conceived by reference to the above-mentioned linear and / or branched configurations.

[0717] As can be seen from the claims and the patent specification, the scope of the present application covers linear or branched structures, and there is no limit on the number of individual ligands that may be present. In addition, the recipient will also be aware that there are many structures that can be used as linker moieties based on the state of the art and the expertise of oligonucleoside chemists.

[0718] The remainder of the total linker described in the claims and the rest of the patent specification (except the linker moiety) is shown by the chemical components in its formula (I), which the inventors believe is particularly unique to this application. However, more generally, these chemical components can be described as "tether moieties" as described above, where the "tether moiety" is part of the total linker, including the atomic group between Z (i.e., the oligonucleoside moiety) and the linker moiety shown in formula (I).

[0719] Tethering moiety of Formula I

[0720] With reference to Formula (I), the "tethering moiety" comprises the group of atoms between Z (ie, the oligonucleotide moiety) and the linker moiety.

[0721] In some embodiments, each occurrence of R1 is hydrogen. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl.

[0722] In some embodiments, R2 is hydroxy. 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.

[0723] In some embodiments, X1 is methylene. In some embodiments, X1 is oxygen. In some embodiments, X1 is sulfur.

[0724] In some embodiments, X2 is methylene. In some embodiments, X2 is oxygen. In some embodiments, X2 is sulfur.

[0725] In some embodiments, m=3.

[0726] In some embodiments, n=6.

[0727] In some embodiments, X1 is oxygen and X2 is methylene. In some embodiments, X1 and X2 are both methylene.

[0728] 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.

[0729] In some embodiments, R1 is hydrogen at each occurrence, n=6, m=3, R2 is fluoro, X2 is methylene, v=1, t=1, s=1, X1 is methylene, q=1 and r=2.

[0730] Therefore, in some embodiments, the exemplary compounds of the present application include the following structures:

[0731]

[0732] In some embodiments, R1 is hydrogen at each occurrence, n=6, m=3, R2 is fluoro, X2 is methylene, v=1, t=1, s=1, X1 is oxygen, q=1 and r=2.

[0733] Therefore, in some embodiments, the exemplary compounds of the present application include the following structures:

[0734]

[0735] Replacement tether section

[0736] During the synthesis of the compounds of the present application, alternative tethering moiety structures may arise. In some embodiments, the alternative tethering moieties have one or more changes in the tethering moiety of the overall linker compared to the tethering moieties described anywhere herein.

[0737] In some embodiments, the alternative tethering moiety is a compound of formula (I) as described anywhere herein, wherein R2 is hydroxy.

[0738] In some embodiments, R1 is hydrogen at each occurrence, n=6, m=3, R2 is hydroxy, X2 is methylene, v=1, t=1, s=1, X1 is methylene, q=1 and r=2.

[0739] Therefore, in some embodiments, the compounds of the present application include the following structure:

[0740]

[0741] In some embodiments, R1 is hydrogen at each occurrence, n=6, m=3, R2 is hydroxy, X2 is methylene, v=1, t=1, s=1, X1 is oxygen, q=1 and r=2.

[0742] Therefore, in some embodiments, the compounds of the present application include the following structure:

[0743]

[0744] Connector

[0745] With respect to Formula (I), the "linker moiety" shown in Formula (I) includes the group of atoms positioned between the tether moiety described anywhere herein and the ligand moiety described anywhere herein.

[0746] In some embodiments:

[0747]

[0748] For example, as described anywhere herein, formula (I) may be any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa):

[0749]

[0750] in:

[0751] A I is hydrogen, or a suitable hydroxy protecting group;

[0752] a is an integer of 2 or 3; and

[0753] b is an integer from 2 to 5; or

[0754]

[0755] in:

[0756] A I is hydrogen or a suitable hydroxy protecting group;

[0757] a is an integer of 2 or 3; and

[0758] c and d are independently an integer from 1 to 6; or

[0759]

[0760] in:

[0761] A I is hydrogen or a suitable hydroxy protecting group;

[0762] a is an integer of 2 or 3; and

[0763] e is an integer from 2 to 10.

[0764] In some embodiments, for example, the moiety described by formula (I): It is formula (VIa):

[0765]

[0766] in:

[0767] A I is hydrogen or a suitable hydroxy protecting group;

[0768] a is 3; and

[0769] b is the integer 3.

[0770] In some embodiments, for example, a moiety described in Formula (I) as described anywhere herein:

[0771] It is formula (VII):

[0772]

[0773] in:

[0774] A I It is hydrogen;

[0775] a is an integer of 2 or 3, preferably 3.

[0776] Other exemplary compounds of the present application comprise a "linker moiety" such as that described by formula (I*), which is part of an overall "linker".

[0777]

[0778] in

[0779] r and s are independently integers selected from 1-16; and

[0780] Z is an oligonucleoside moiety.

[0781] As will be further understood in the art, the exemplary compounds of the present application include an overall linker located between the oligonucleoside portion and the ligand portion of these compounds. The overall linker thereby "connects" the oligonucleoside portion and the ligand portion to each other.

[0782] The total linker is generally conceptualized as being composed of one or more linker building blocks. For example, there is a linker portion, described as a "linker portion", such as shown in formula (I*), which is located near the ligand portion, and the ligand portion is usually directly or indirectly connected to the oligonucleoside portion through a branch point. The linker portion described by formula (I*) is also generally referred to as the "ligand arm (arm)" of the total linker. There may also be (but not always) another connecting portion between the oligonucleoside portion and the branch point, which is generally referred to as the "tethering portion" of the total linker, which "tethers" the oligonucleoside portion to the rest of the coupling compound. This "ligand arm" and / or "linker portion" and / or "tethering portion" can be conceived by reference to the above-mentioned linear and / or branched configurations.

[0783] As can be seen from the claims and the patent specification, the scope of the present application covers linear or branched structures, and there is no limit on the number of individual ligands that may be present. In addition, the recipient will also be aware that there are many structures that can be used as linker moieties based on the state of the art and the expertise of oligonucleoside chemists.

[0784] The remainder of the total linker described in the claims and the rest of the patent specification (except the linker moiety) is shown by the chemical components in its formula (I), which the inventors believe is particularly unique to this application. However, more generally, these chemical components can be described as "tether moieties" as described above, where the "tether" is part of the total linker, including the atomic group between Z (i.e., the oligonucleoside moiety) and the linker moiety shown in formula (I).

[0785] Tether part

[0786] With respect to formula (I*), the "tethering moiety" comprises the group of atoms between Z (ie, the oligonucleotide moiety) and the linker moiety.

[0787] In some embodiments, s is an integer selected from 4 to 12. In some embodiments, s is 6.

[0788] In some embodiments, r is an integer selected from 4 to 14. In some embodiments, r is 6. In some embodiments, r is 12.

[0789] In some embodiments, r is 12 and s is 6.

[0790] Therefore, in some embodiments, the exemplary compounds of the present application include the following structures:

[0791]

[0792] In some embodiments, r is 6 and s is 6

[0793] Therefore, in some embodiments, the exemplary compounds of the present application include the following structures:

[0794]

[0795] Connector

[0796] With respect to Formula (I*), the "linker moiety" shown in Formula (I*) includes the group of atoms positioned between the tether moiety described anywhere herein and the ligand moiety described anywhere herein.

[0797] In some embodiments:

[0798] For example, as described in formula (I*) described anywhere herein

[0799]

[0800] is any one of formula (IV*), (V*) or (VI*), preferably formula (IV*):

[0801]

[0802] in:

[0803] A I is hydrogen, or a suitable hydroxy protecting group;

[0804] a is the integer 2 or 3; and

[0805] b is an integer from 2 to 5; or

[0806]

[0807] in:

[0808] A I is hydrogen, or a suitable hydroxy protecting group;

[0809] a is the integer 2 or 3; and

[0810] c and d are independently an integer from 1 to 6; or

[0811]

[0812] in:

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

[0814] a is the integer 2 or 3; and

[0815] e is an integer from 2 to 10.

[0816] In some embodiments, for example, the moiety described by formula (I):

[0817] It is formula (VIa*):

[0818]

[0819] in:

[0820] A I is hydrogen, or a suitable hydroxy protecting group;

[0821] a is 3; and

[0822] b is the integer 3.

[0823] In some embodiments, for example, a moiety described in Formula (I) as described anywhere herein: It is formula (VII*):

[0824]

[0825] in:

[0826] A I It is hydrogen;

[0827] a is an integer of 2 or 3, preferably 3.

[0828] In some embodiments, a=2. In some embodiments, a=3. In some embodiments, b=3.

[0829] Vectors and cells

[0830] In one aspect, the present application provides a cell containing a nucleic acid (eg, an inhibitory RNA [RNAi] described herein).

[0831] In one aspect, the present application provides a cell comprising the vector described herein.

[0832] In one aspect, the present application provides a vector comprising an oligonucleotide inhibitor, such as an iRNA, such as a siRNA.

[0833] Pharmaceutically acceptable compositions

[0834] In one aspect, the present application provides a pharmaceutical composition for inhibiting the expression of a target gene, the composition comprising an inhibitor, such as an oligomer disclosed herein, such as a nucleic acid.

[0835] A pharmaceutically acceptable composition may comprise an excipient and / or a carrier.

[0836] Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) 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) buffers, 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 buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) fillers, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances used in pharmaceutical preparations.

[0837] Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium carboxymethyl starch, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0838] Pharmaceutically acceptable organic or inorganic excipients, suitable for non-parenteral administration, which do not react harmfully with nucleic acids, can also be used to prepare the compositions of the present application. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, straight-chain starch, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, etc.

[0839] Preparations for topical administration of nucleic acid may include sterile and non-sterile aqueous solutions, common solvents such as non-aqueous solutions in alcohol, or solutions of nucleic acid in liquid or solid oil bases. The solution may also contain buffer, diluent and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients may be used, suitable for non-parenteral administration, which will not have a deleterious reaction with the nucleic acid used.

[0840] In one embodiment, the nucleic acid or composition is administered in an unbuffered solution. In certain embodiments, the unbuffered solution is saline or water. In other embodiments, the nucleic acid (e.g., RNAi reagent) is administered in a buffered solution. In these embodiments, the buffered solution may include acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. For example, the buffered solution may be phosphate buffered saline (PBS).

[0841] dose

[0842] The pharmaceutical composition of the application can be administered with enough inhibition of gene expression or change target expression or function.Usually, when compositions comprise nucleic acid, the suitable dosage of nucleic acid (such as siRNA) of the application will be in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, usually in the range of about 1-50 milligrams per kilogram of body weight per day.Usually, the suitable dosage of nucleic acid (such as siRNA of the application) will be in the range of about 0.1mg / kg to about 5.0mg / kg, for example, about 0.3mg / kg and about 3.0mg / kg.

[0843] Repeated dosage regimens can include regular administration of therapeutic amounts of nucleic acids, such as siRNA, such as every other day or once a year. In some embodiments, the nucleic acid (e.g., siRNA) is administered about once a month to about once a quarter (i.e., administered about every three months).

[0844] In some embodiments, the nucleic acid (e.g., siRNA) agent is administered at a dosage of about 0.01 mg / kg to about 10 mg / kg, or about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered at a dosage of about 10 mg / kg to about 30 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered at a dosage 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 some embodiments, the nucleic acid (e.g., siRNA) agent is administered once a week, monthly, every two months, or quarterly (i.e., once every three months) at a dosage of about 0.1 mg / kg to about 5.0 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered to a subject once a week. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered to a subject once a month. In some embodiments, the nucleic acid (e.g., siRNA) agent is administered to a subject once a quarterly (i.e., every three months).

[0845] After the initial treatment regimen, the dosing frequency can be reduced. For example, after three months of weekly or biweekly dosing, dosing can be repeated monthly for six months or a year; or longer.

[0846] The pharmaceutical composition can be administered once a day, or in one day with two, three or more divided doses at appropriate intervals, and can even be continuously infused or administered by controlled release formulations. In this case, in order to reach the daily total dose, the nucleic acid (such as siRNA) contained in each divided dose must be reduced accordingly. The dosage unit can also be configured to send in a few days, such as using conventional sustained-release formulations, which provide the sustained release of nucleic acid (such as siRNA) in a few days. Sustained-release formulations are well-known in the art, and are particularly suitable for delivering reagents at specific locations, such as can be used together with the reagent of the application. In this embodiment, the dosage unit comprises the corresponding multiple of the daily dose.

[0847] In other embodiments, the pharmaceutical composition of a single dose can have a long-acting effect, so 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 application, a single dose of the pharmaceutical composition of the application is administered once a week. In other embodiments of the application, a single dose of the pharmaceutical composition of the application is administered once every two months. In some embodiments, the siRNA is administered once about once a month to about once per quarter (i.e., about once every three months), or even administered once every 6 months or 12 months.

[0848] As known in the art, estimates of effective dosages and in vivo half-lives of individual nucleic acids (eg, siRNAs) encompassed by the present application can be made using conventional methods or based on in vivo testing using appropriate animal models.

[0849] The pharmaceutical composition of the present application can be administered in a variety of ways, depending on whether local or systemic treatment is needed and depending on the area to be treated. Administration can be local (e.g., by transdermal patch), pulmonary, such as by inhalation or blowing into powder or aerosol, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, such as by implantation device; or intracranial administration, such as by intracerebral parenchyma, intrathecal or intraventricular administration. In some preferred embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by subcutaneous injection.

[0850] In one embodiment, the nucleic acid (eg, siRNA) agent is administered subcutaneously to the subject.

[0851] Inhibitors, such as nucleic acids, such as siRNA, can be delivered in a manner that targets specific tissues, such as, in particular, hepatocytes.

[0852] Methods for inhibiting gene expression or inhibiting target expression or function

[0853] The present application also provides methods for inhibiting gene expression in cells and methods for inhibiting expression and / or function of other target molecules. The method comprises contacting the cell with a nucleic acid (e.g., an siRNA agent, e.g., a double-stranded siRNA) of the present application in an amount sufficient to effectively inhibit the expression of the gene in the cell, thereby inhibiting the expression of the gene in the cell. In a preferred embodiment, the gene is ZPI.

[0854] The contact of cells with inhibitors (e.g., nucleic acids, such as siRNA, such as double-stranded siRNA agents) can be carried out in vitro or in vivo. Contacting cells with inhibitor nucleic acids (e.g., siRNA) in vivo includes contacting cells or cell groups in a subject (e.g., a human subject) with nucleic acids (e.g., siRNA). Combinations of in vitro and in vivo methods of cell contact are also possible. As described above, contacting cells can be direct or indirect. In addition, contacting cells can be achieved by targeting ligand moieties (including any ligand moieties described herein or known in the art). In a preferred embodiment, the targeting ligand moiety is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to the target site.

[0855] The term "inhibit," as used herein, is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any degree of inhibition.

[0856] In some embodiments of the methods of the present application, the expression or activity of a gene or an inhibitory target is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or below the level of detection determined, preferably when measured by qPCR as described herein and / or when siRNA is introduced into target cells by transfection. In some embodiments, the methods include clinically relevant inhibition of target gene expression, for example, as demonstrated by clinically relevant results after treating a subject with an agent that reduces target gene expression and / or target activity.

[0857] In some embodiments, when transfected into cells, the nucleic acids described herein inhibit the expression of the ZPI gene with an IC50 value of less than 2500 pM, 2400 pM, 2300 pM, 2200 pM, 2100 pM, 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM, preferably as determined by qPCR, more preferably as determined by reverse transcriptase (RT)-qPCR. As described herein.

[0858] In a preferred embodiment, when transfected into a cell, the nucleic acids described herein inhibit the expression of the ZPI gene with an IC50 value of less than 2500 pM. In a more preferred embodiment, when transfected into a cell, the nucleic acids described herein inhibit the expression of the ZPI gene with an IC50 value of less than 1000 pM. In a more preferred embodiment, when transfected into a cell, the nucleic acids described herein inhibit the expression of the ZPI gene with an IC50 value of less than 500 pM. In a most preferred embodiment, when transfected into a cell, the nucleic acids described herein inhibit the expression of the ZPI gene with an IC50 value of less than 100 pM.

[0859] Inhibition of ZPI gene expression can be quantified by the following method:

[0860] Huh7 cells (a cell line derived from human hepatocytes, obtained from the JCRB cell bank) can be cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO2. Cells can then be transfected with siRNA duplexes targeting ZPI mRNA or negative control siRNA (siRNA-control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 794), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 790)) in a final duplex concentration range of 20nM-1pM using 10x3-fold serial dilutions. Transfection can be performed by adding 9.7μL Opti-MEM (ThermoFisher) plus 0.3μL Lipofectamine RNAiMAX (ThermoFisher) to 10μL of each siRNA duplex. The mixture can be incubated at room temperature for 15 minutes, and then 100 μL of complete growth medium containing 20,000 Huh7 cells is added. The cells can be incubated at 37°C / 5% CO2 for 24 hours, and then total RNA is purified using the RNeasy 96 kit (Qiagen). In a single experiment, each duplex can be tested by transfection in duplicate in a well.

[0861] cDNA synthesis can be performed using the FastQuant RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the FastStart Universal Probe Master Kit (Roche) with specific primers for human ZPI (Hs01547819_m1) and human GAPDH (Hs02786624_g1).

[0862] qPCR can be performed in duplicate on cDNA from each well and the average cycle threshold (Ct) calculated. Relative expression of ZPI relative to untreated cells can be calculated from the average Ct values ​​using the comparative Ct method, normalized to GAPDH. Maximum percent inhibition of ZPI expression and IC50 values ​​can be calculated using a four-parameter (variable slope) model in GraphPad Prism 9.

[0863] Alternatively or additionally, inhibition of ZPI gene expression may be characterized by a decrease in the mean relative expression of the ZPI gene.

[0864] In some embodiments, when cells are transfected with 0.1 nM of a nucleic acid described herein, the average relative expression of ZPI is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5 or 0.4, as described herein, preferably when measured by qPCR, more preferably by reverse transcriptase (RT)-qPCR.

[0865] In some embodiments, when cells are transfected with 5 nM of a nucleic acid described herein, the average relative expression of ZPI is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3, as described herein, preferably as determined by qPCR, more preferably as determined by reverse transcriptase (RT)-qPCR.

[0866] The average relative expression of ZPI genes can be quantified by:

[0867] Huh7 cells (a cell line derived from human hepatocytes, obtained from the JCRB cell bank) can be cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO2. Cells can be transfected with siRNA duplexes targeting ZPI mRNA or negative control siRNA (siRNA-control; sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 794), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 790)) at a final duplex concentration range of 5 nM and 0.1 pM. Transfection can be performed by adding 9.7 μL Opti-MEM (Thermo Fisher) plus 0.3 μL Lipofectamine RNAiMAX (Thermo Fisher) to 10 μL of each siRNA duplex. The mixture can be incubated at room temperature for 15 minutes, and then 100 μL of complete growth medium containing 20,000 Huh7 cells is added. The cells can be incubated at 37°C / 5% CO2 for 24 hours, and then total RNA is purified using the RNeasy 96 kit (Qiagen). In two independent experiments, each duplex can be tested by transfection in duplicate in the well.

[0868] cDNA synthesis can be performed using the FastQuant RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using the FastStart Universal Probe Master Kit (Roche) with specific primers for human ZPI (Hs01547819_m1) and human GAPDH (Hs02786624_g1).

[0869] qPCR can be performed in duplicate on cDNA from each well, and the average Ct is calculated. The relative expression of ZPI relative to untreated cells can be calculated from the average Ct values ​​using the comparative Ct (ΔΔCt) method, normalized to GAPDH.

[0870] Inhibition of gene expression may be manifested as a decrease in the amount of mRNA of the target gene of interest compared to a suitable control. Inhibition of target function may be manifested as a decrease in target activity compared to a suitable control.

[0871] In other embodiments, inhibition of gene or other target expression can be assessed based on a reduction in a parameter functionally related to gene expression, such as protein expression or a signaling pathway.

[0872] Method for treating or preventing diseases related to target gene expression / functional expression

[0873] The application also provides a method for reducing or inhibiting gene expression in a cell or reducing the expression or function of a target using a nucleic acid (e.g., siRNA of the application) or a composition containing a nucleic acid (e.g., siRNAs of the application). The method comprises contacting a cell with a nucleic acid (e.g., dsiRNA of the application) and keeping the cell for a sufficiently long time to obtain degradation of the mRNA transcript of the gene, thereby inhibiting the expression of the gene in the cell. The reduction in gene expression or function of the target can be assessed by any method known in the art. In a preferred embodiment, the gene is ZPI.

[0874] In the methods of the present application, the cells may be contacted in vitro or in vivo, ie, the cells may be within the body of a subject.

[0875] Cells suitable for treatment using the methods of the present application can be any cells that express a target gene or target associated with a disease.

[0876] The in vivo method of the present application may include administering to a subject a composition containing a nucleic acid (e.g., iRNA) of the present application, wherein the nucleic acid (e.g., siRNA) includes a nucleotide sequence complementary to at least a portion of an RNA transcript of a mammalian gene to be treated, or a nucleotide sequence complementary to another nucleic acid whose expression and / or function is associated with a disease.

[0877] The present application also provides a method for treating a subject in need. The treatment method of the present application includes administering a nucleic acid (e.g., siRNA) of the present application or a pharmaceutical composition comprising a nucleic acid targeting a gene to a subject (e.g., a subject who will benefit from reducing or inhibiting the expression of a gene and / or the expression and / or function of a target) in a therapeutically effective amount (e.g., a nucleic acid targeting a gene, such as siRNA).

[0878] The nucleic acid of the present application, such as siRNA, can be administered as "free" nucleic acid or "free" siRNA, and is administered without a pharmaceutical composition. The naked nucleic acid can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH value and osmotic pressure of the buffer solution can be adjusted to make it suitable for administration to a subject.

[0879] Alternatively, the nucleic acids of the present application, such as siRNA, can be administered as a pharmaceutical composition, such as a dsiRNA liposome formulation.

[0880] In one embodiment, the method includes administering a composition characterized herein so that the expression of the target gene is reduced, for example, for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32 or about 36 hours. In one embodiment, the expression of the target gene is reduced in an extended duration, for example, at least about two, three, four days or longer, for example, about one week, two weeks, three weeks or four weeks or longer, for example, about 1 month, 2 months or 3 months.

[0881] A therapeutic amount of a nucleic acid, such as siRNA, can be administered to a subject, for example, from about 0.01 mg / kg to about 200 mg / kg.

[0882] Nucleic acids (e.g., siRNA) can be administered periodically over a period of time by intravenous infusion. In some embodiments, after the initial treatment regimen, the frequency of administration can be reduced. The administration of siRNA can reduce the gene product level of the target gene, for example, by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% in patient cells or tissues, or to a level below the detection level of the detection method used. In some embodiments, administration results in a reduction in at least one sign or symptom of a clinically stable or preferably clinically relevant gene-related disease.

[0883] Alternatively, nucleic acid (e.g., siRNA) can be administered by subcutaneous injection, i.e., subcutaneous injection. One or more injections can be used to deliver a desired daily dose of nucleic acid, e.g., siRNA, to a subject. Injection can be repeated over a period of time. Administration can be repeated regularly. In some embodiments, after the initial treatment regimen, the administration frequency can be reduced. Repeated dosing regimens can include nucleic acid for regular administration of a therapeutic amount, e.g., every other day or once a year. In some embodiments, the nucleic acid is administered about once a month to about once a quarter (i.e., about once every three months).

[0884] In one aspect, the present application can be applied to the compounds, methods, compositions or uses of the following statements numbered 1-101 (wherein any structural formula mentioned in statements 1-101 refers only to those structural formulas defined in statements 1-110. These structural formulas are as follows Figure 6 (shown)

[0885] 1. A compound comprising the following structure:

[0886]

[0887] in:

[0888] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[0889] R2 is selected from the group consisting of hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[0890] X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;

[0891] m is an integer from 1 to 6;

[0892] n is an integer from 1 to 10;

[0893] q, r, s, t, v are independently integers from 0 to 4, provided that:

[0894] (i) q and r cannot be zero at the same time; and

[0895] (ii) s, t and v cannot be 0 at the same time;

[0896] Z is an oligonucleoside moiety.

[0897] 2. A compound according to statement 1, wherein R1 at each occurrence is hydrogen.

[0898] 3. A compound according to claim 1, wherein R1 is methyl.

[0899] 4. A compound according to statement 1, wherein R1 is ethyl.

[0900] 5. A compound according to any one of clauses 1 to 4, wherein R2 is hydroxy.

[0901] 6. A compound according to any one of clauses 1 to 4, wherein R2 is halogen.

[0902] 7. A compound according to clause 6, wherein R2 is fluoro.

[0903] 8. A compound according to clause 6, wherein R2 is chloro.

[0904] 9. A compound according to clause 6, wherein R2 is bromine.

[0905] 10. The compound according to clause 6, wherein R2 is iodine.

[0906] 11. The compound according to clause 6, wherein R2 is nitro.

[0907] 12. A compound according to any one of clauses 1 to 11, wherein X1 is methylene.

[0908] 13. A compound according to any one of clauses 1 to 11, wherein X1 is oxygen.

[0909] 14. A compound according to any one of clauses 1 to 11, wherein X1 is sulfur.

[0910] 15. A compound according to any one of clauses 1 to 14, wherein X2 is methylene.

[0911] 16. A compound according to any one of clauses 1 to 15, wherein X2 is oxygen.

[0912] 17. A compound according to any one of clauses 1 to 16, wherein X2 is sulfur.

[0913] 18. A compound according to any one of clauses 1-17, wherein m=3.

[0914] 19. A compound according to any one of clauses 1-18, wherein n=6.

[0915] 20. The compound according to clauses 13 and 15, wherein X1 is oxygen, X2 is methylene, preferably wherein:

[0916] q=1, r=2, s=1, t=1, v=1.

[0917] 21. A compound according to clauses 12 and 15, wherein X1 and X2 are both methylene, preferably wherein:

[0918] q=1, r=3, s=1, t=1, v=1.

[0919] 22. A compound according to any one of clauses 1 to 21, wherein Z is:

[0920]

[0921] in:

[0922] Z1, Z2, Z3, Z4 at each occurrence are independently oxygen or sulfur; and

[0923] The bond between P and Z2 and one bond between P and Z3 is a single bond and the other bond is a double bond.

[0924] 23. A compound according to statement 22, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0925] 24. A compound according to statement 23, wherein the RNA compound comprises an 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 each of the first strand and the second strand has a 5' and a 3' end.

[0926] 25. The compound according to clause 24, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[0927] 26. The compound according to clause 24, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[0928] 27. A compound of formula (II):

[0929]

[0930] 28. A compound of formula (III):

[0931]

[0932] 29. A compound according to statement 27 or 28, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 5' end of its second strand.

[0933] 30. A composition comprising a compound of formula (II) as defined in statement 27, and a compound of formula (III) as defined in statement 28, optionally subject to statement 29.

[0934] 31. The composition according to clause 30, wherein said compound of formula (III) as defined in clause 28 is present in an amount of 10-15% by weight of said composition.

[0935] 32. A compound of formula (IV):

[0936]

[0937] 33. A compound of formula (V):

[0938]

[0939] 34. A compound according to statement 32 or 33, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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.

[0940] 35. A composition comprising a compound of formula (IV) as defined in clause 32, and a compound of formula (V) as defined in clause 33, optionally subject to clause 34.

[0941] 36. The composition according to clause 35, wherein said compound of formula (V) as defined in clause 33 is present in an amount of 10-15% by weight of said composition.

[0942] 37. A compound as defined in any one of clauses 1-29 or 32-34, 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.

[0943] 38. A compound according to clause 37, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0944] 39. A compound according to any one of clauses 1-29, or 32-34, or 37-38, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0945] 40. A compound according to statement 39, wherein the one or more degradation protection parts are not present at the end of the oligonucleoside chain carrying the ligand portion, and / or wherein the one or more degradation protection parts are selected from thiophosphate internucleoside bonds, dithiophosphate internucleoside bonds and reverse abasic nucleosides, wherein the reverse abasic nucleosides are present at the distal end of the chain carrying the ligand portion.

[0946] 41. A compound according to any one of statements 1-29, or 32-34, or 37-40, wherein the ligand moiety as described by formula (I) in statement 1 comprises one or more ligands.

[0947] 42. The compound according to clause 41, wherein the ligand moiety as described in clause 1 as formula (I) comprises one or more carbohydrate ligands.

[0948] 43. The compound according to clause 42, wherein the one or more carbohydrates can be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide.

[0949] 44. A compound according to statement 43, wherein the one or more carbohydrates comprises one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties and / or one or more mannose moieties.

[0950] 45. A compound according to clause 44, wherein said one or more carbohydrates comprises one or more N-acetylgalactosamine moieties.

[0951] 46. ​​A compound according to clause 45, comprising two or three N-acetylgalactosamine moieties.

[0952] 47. A compound according to clauses 41-46, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[0953] 48. A compound according to clause 47, wherein the one or more ligands are attached in a bifurcated or trifurcated branched configuration.

[0954] 49. A compound according to clauses 46-48, wherein the linker described in formula (I) in clause 1:

[0955]

[0956] is any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa):

[0957]

[0958] in:

[0959] A I is hydrogen, or a suitable hydroxy protecting group;

[0960] a is an integer 2 or 3; and

[0961] b is an integer from 2 to 5; or

[0962]

[0963] in:

[0964] A I is hydrogen or a suitable hydroxy protecting group;

[0965] a is an integer 2 or 3; and

[0966] c and d are independently an integer from 1 to 6; or

[0967]

[0968] in:

[0969] A I is hydrogen or a suitable hydroxy protecting group;

[0970] a is an integer 2 or 3; and

[0971] e is an integer from 2 to 10.

[0972] 50. A compound according to clauses 46-48, wherein the linker described in formula (I) in clause 1:

[0973]

[0974] It is formula (VII)

[0975]

[0976] in:

[0977] A I It is hydrogen;

[0978] a is an integer 2 or 3.

[0979] 51. A compound according to clause 49 or 50, wherein a=2.

[0980] 52. A compound according to clause 49 or 50, wherein a=3.

[0981] 53. The compound according to clause 49, wherein b=3.

[0982] 54. A compound of formula (VIII):

[0983]

[0984] 55. A compound of formula (IX):

[0985]

[0986] 56. A compound according to statement 54 or 55, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 connected to an adjacent phosphate group at the 5' end of its second strand.

[0987] 57. A composition comprising a compound of formula (VIII) as defined in clause 54, and a compound of formula (IX) as defined in clause 55, optionally subject to clause 56.

[0988] 58. The composition according to clause 57, wherein said compound of formula (IX) as defined in clause 55 is present in an amount of 10-15% by weight of said composition.

[0989] 59. A compound of formula (X):

[0990]

[0991] 60. A compound of formula (XI):

[0992]

[0993] 61. A compound according to statement 59 or 60, wherein the oligonucleoside comprises an RNA duplex comprising a first 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 connected to an adjacent phosphate group at the 3' end of its second strand.

[0994] 62. A composition comprising a compound of formula (X) as defined in clause 59, and a compound of formula (XI) as defined in clause 60, optionally subject to clause 61.

[0995] 63. The composition according to clause 62, wherein the compound of formula (XI) as defined in clause 60 is present in an amount of 10-15% by weight of the composition.

[0996] 64. A compound as defined in any one of clauses 54 to 63, 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.

[0997] 65. A compound according to clause 64, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[0998] 66. A compound according to any one of clauses 54-65, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0999] 67. A compound according to statement 66, wherein the one or more degradation protection moieties are not present at the end of the oligonucleoside chain carrying the ligand portion, and / or wherein the one or more degradation protection moieties are selected from thiophosphate internucleoside linkages, dithiophosphate internucleoside linkages and reverse abasic nucleosides, wherein the reverse abasic nucleosides are present at the distal end of the chain carrying the ligand portion, as shown in any of formulas (VIII), (IX), (X) or (XI) of any of statements 54, 55, 59 or 60.

[1000] 68. A method for preparing a compound according to any one of clauses 1-29, 32-34, 37-56, 59-61 and 64-67, and / or a composition according to any one of clauses 30, 31, 35, 36, 57, 58, 62, 63, comprising reacting compounds of formula (XII) and (XIII):

[1001]

[1002] in:

[1003] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1004] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[1005] X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;

[1006] m is an integer from 1 to 6;

[1007] n is an integer from 1 to 10;

[1008] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1009] (i) q and r cannot be zero at the same time; and

[1010] (ii) s, t and v cannot all be 0 at the same time;

[1011] Z is an oligonucleoside moiety,

[1012] and, where appropriate, deprotecting the ligand and / or annealing the second strand of the oligonucleotide moiety.

[1013] 69. The method according to clause 68, wherein the compound of formula (XII) is prepared by reacting a compound of formula (XIV) and a compound of formula (XV):

[1014]

[1015] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1016] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[1017] X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;

[1018] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1019] (i) q and r cannot be zero at the same time; and

[1020] (ii) s, t and v cannot all be 0 at the same time;

[1021] Z is an oligonucleoside moiety.

[1022] 70. The method of claim 68, preparing a compound according to any one of claims 20, 25, 27, 29, 54, 56, and / or a composition according to any one of claims 30, 31, 57, 58, wherein:

[1023] The compound of formula (XII) is formula (XIIa):

[1024]

[1025] And the compound of formula (XIII) is formula (XIIIa):

[1026]

[1027] wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 5' end of the second strand thereof.

[1028] 71. A method according to claim 68, preparing a compound according to any one of claims 20, 25, 28, 29, 55, 56, and / or a composition according to any one of claims 30, 31, 57, 58, wherein:

[1029] The compound of formula (XII) is formula (XIIb):

[1030]

[1031] And the compound of formula (XIII) is formula (XIIIa)

[1032]

[1033] wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 5' end of the second strand thereof.

[1034] 72. A method according to claim 68, preparing a compound according to any one of claims 21, 26, 32, 34, 59, 61, and / or a composition according to any one of claims 35, 36, 62, 63, wherein:

[1035] The compound of formula (XII) is formula (XIIc):

[1036]

[1037] And the compound of formula (XIII) is formula (XIIIa):

[1038]

[1039] wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 the second strand thereof.

[1040] 73. A method according to claim 68, preparing a compound according to any one of claims 21, 26, 33, 34, 60, 61, and / or a composition according to any one of claims 35, 36, 62, 63, wherein:

[1041] The compound of formula (XII) is formula (XIId):

[1042]

[1043] And the compound of formula (XIII) is formula (XIIIa)

[1044]

[1045] wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 the second strand thereof.

[1046] 74. The method according to any one of clauses 70 to 73, wherein:

[1047] The compound of formula (XIIIa) is formula (XIIIb):

[1048]

[1049] 75. The method of clause 69 as appended to clauses 70-73, wherein:

[1050] The compound of formula (XIV) is formula (XIVa) or formula (XIVb)

[1051]

[1052] and the compound of formula (XV) is formula (XVa) or formula (XVb)

[1053]

[1054] The oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5' and a 3' end, and wherein (i) the RNA duplex is linked to adjacent phosphate groups in formula (XVa) at the 5' end of the second strand, or (ii) the RNA duplex is linked to adjacent phosphate groups in formula (XVb) at the 3' end of the second strand.

[1055] 76. A compound of formula (XII):

[1056]

[1057] in,

[1058] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1059] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[1060] X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur;

[1061] q, r, s, t, v are independently integers from 0 to 4, provided that:

[1062] (i) q and r cannot be zero at the same time; and

[1063] (ii) s, t and v cannot all be 0 at the same time;

[1064] Z is an oligonucleoside moiety.

[1065] 77. A compound of formula (XIIa):

[1066]

[1067] 78. A compound of formula (XIIb):

[1068]

[1069] 79. A compound of formula (XIIc):

[1070]

[1071] 80. A compound of formula (XIId)

[1072]

[1073] 81. A compound having the formula (XIII)

[1074]

[1075] in:

[1076] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1077] m is an integer from 1 to 6;

[1078] n is an integer from 1 to 10.

[1079] 82. A compound having the formula (XIIIa)

[1080]

[1081] 83. A compound having the formula (XIIIb)

[1082]

[1083] 84. A compound of formula (XIV)

[1084]

[1085] in:

[1086] R1 is selected from the group consisting of hydrogen, methyl and ethyl;

[1087] R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro;

[1088] X1 is selected from the group consisting of methylene, oxygen and sulfur;

[1089] s, t, and v are independently integers from 0 to 4, provided that s, t, and v cannot all be 0 at the same time.

[1090] 85. A compound of formula (XIVa)

[1091]

[1092] 86. A compound of formula (XIVb)

[1093]

[1094] 87. A compound of formula (XV)

[1095]

[1096] in:

[1097] R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl;

[1098] X1 is selected from the group consisting of methylene, oxygen and sulfur;

[1099] q and r are independently integers from 0 to 4, provided that q and r cannot be 0 at the same time;

[1100] Z is an oligonucleoside moiety.

[1101] 88. A compound of formula (XVa)

[1102]

[1103] 89. A compound of formula (XVb)

[1104]

[1105] 90. Use of a compound according to any one of clauses 76, 81-84, 87 for the preparation of a compound according to any one of clauses 1-29, 32-34, 37-56, 59-61 and 64-67, and / or a composition according to any one of clauses 30, 31, 35, 36, 57, 58, 62 and 63.

[1106] 91. Use of a compound according to clause 85 for preparing a compound according to any one of clauses 1-29, 32-34, 37-56, 59-61 and 64-67, and / or a composition according to any one of clauses 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2 = F.

[1107] 92. Use of a compound according to clause 86 for preparing a compound according to any one of clauses 1-29, 32-34, 37-56, 59-61 and 64-67, and / or a composition according to any one of clauses 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2 = OH.

[1108] 93. Use of a compound according to clause 77 for preparing a compound according to any one of clauses 20, 25, 27, 29, 54, 56, and / or a composition according to any one of clauses 30, 31, 57, 58.

[1109] 94. Use of a compound according to clause 78 for preparing a compound according to any one of clauses 20, 25, 28, 29, 55, 56, and / or a composition according to any one of clauses 30, 31, 57, 58.

[1110] 95. Use of a compound according to clause 79 for preparing a compound according to any one of clauses 21, 26, 32, 34, 59, 61, and / or a composition according to any one of clauses 35, 36, 62, 63.

[1111] 96. Use of a compound according to clause 80 for preparing a compound according to any one of clauses 21, 26, 33, 34, 59, 61, and / or a composition according to any one of clauses 35, 36, 62, 63.

[1112] 97. Use of a compound according to clause 88 for preparing a compound according to any one of clauses 20, 25, 27-29, 54-56, and / or a composition according to any one of clauses 30, 31, 57, 58.

[1113] 98. Use of a compound according to clause 89 for preparing a compound according to any one of clauses 21, 26, 32-34, 59-61, and / or a composition according to any one of clauses 35, 36, 62, 63.

[1114] 99. A compound or composition obtained or obtainable by a method according to any one of clauses 68-75.

[1115] 100. A pharmaceutical composition comprising a compound according to any one of clauses 1-29, 32-34, 37-56, 59-61, and 64-67, and / or a composition according to any one of clauses 30, 31, 35, 36, 57, 58, 62, and 63, and a pharmaceutically acceptable carrier, diluent, or excipient.

[1116] Use of a compound according to any one of clauses 1-29, 32-34, 37-56, 59-61, and 64-67, and / or a composition according to any one of clauses 30, 31, 35, 36, 57, 58, 62 and 63 in therapy.

[1117] In another aspect, the present application can be applied to the compounds, methods, compositions or uses of the following clauses numbered 1-56 (wherein any structural formula mentioned in the clause refers only to those structural formulas defined in clauses 1-56. These structural formulas are as follows Figure 7 shown).

[1118] 1. A compound comprising the following structure:

[1119]

[1120] in

[1121] r and s are independently integers selected from 1-16; and

[1122] Z is an oligonucleoside moiety.

[1123] 2. The compound according to clause 1, wherein s is an integer selected from 4-12.

[1124] 3. The compound according to clause 2, wherein s is 6.

[1125] 4. The compound according to any one of clauses 1-3, wherein r is an integer selected from 4-14.

[1126] 5. The compound according to clause 4, wherein r is 6.

[1127] 6. The compound according to clause 4, wherein r is 12.

[1128] 7. A compound according to clause 5, which compound is subject to clause 3.

[1129] 8. The compound according to clause 6, which compound is subject to clause 3.

[1130] 9. A compound according to any one of clauses 1 to 8, wherein Z is:

[1131]

[1132] in:

[1133] Z1, Z2, Z3, Z4 at each occurrence are independently oxygen or sulfur; and

[1134] One of the bonds between P and Z2, and between P and Z3 is a single bond and the other is a double bond.

[1135] 10. The compound according to any one of clauses 1 to 9, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[1136] 11. A compound according to clause 10, wherein the RNA compound comprises an 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 each of the first strand and the second strand has a 5' and a 3' end.

[1137] 12. The compound according to clause 11, preferably also dependent on clauses 3 and 6, wherein the RNA compound is linked to an adjacent phosphate group at the 5' end of its second strand.

[1138] 13. The compound according to clause 11, preferably also dependent on clauses 3 and 5, wherein the RNA compound is linked to an adjacent phosphate group at the 3' end of its second strand.

[1139] 14. A compound of formula (II*), preferably subject to clause 12:

[1140]

[1141] 15. A compound of formula (III*), preferably subject to clause 13:

[1142]

[1143] 16. A compound as defined in any one of clauses 1 to 15, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2' position, preferably a plurality of riboses modified at the 2' position.

[1144] 17. The compound according to clause 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[1145] 18. A compound according to any one of clauses 1 to 17, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[1146] 19. A compound according to clause 18, wherein the one or more degradation protection moieties are not present at the termini 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 carrying the linker / ligand moiety.

[1147] 20. The compound according to any one of clauses 1 to 19, wherein the ligand moiety as described by formula (I*) in clause 1 comprises one or more ligands.

[1148] 21. The compound according to clause 20, wherein the ligand moiety as described by formula (I*) in clause 1 comprises one or more carbohydrate ligands.

[1149] 22. The compound according to clause 21, wherein the one or more carbohydrates may be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide.

[1150] 23. A compound according to clause 22, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties and / or one or more mannose moieties.

[1151] 24. A compound according to clause 23, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[1152] 25. A compound according to clause 24, comprising two or three N-acetylgalactosamine moieties.

[1153] 26. A compound according to any preceding clause, wherein the one or more ligands are linked in a linear configuration or a branched configuration.

[1154] 27. The compound according to clause 26, wherein the one or more ligands are linked in a bifurcated or trifurcated branched configuration.

[1155] 28. The compound according to clauses 20-27, wherein the linker described in formula (I*) in clause 1 is:

[1156]

[1157] Any one of formula (IV*), (V*) or (VI*), preferably formula (IV*):

[1158]

[1159] in:

[1160] A I is hydrogen, or a suitable hydroxy protecting group;

[1161] a is an integer 2 or 3; and

[1162] b is an integer from 2 to 5; or

[1163]

[1164] in:

[1165] A I is hydrogen or a suitable hydroxy protecting group;

[1166] a is an integer 2 or 3; and

[1167] c and d are independent integers from 1 to 6; or

[1168]

[1169] in:

[1170] A I is hydrogen or a suitable hydroxy protecting group;

[1171] a is an integer 2 or 3; and

[1172] e is an integer from 2 to 10.

[1173] 29. The compound according to any one of clauses 1 to 28, wherein the linker described in formula (I*) in clause 1:

[1174]

[1175] It is formula (VII*):

[1176]

[1177] in:

[1178] A I It is hydrogen;

[1179] a is an integer 2 or 3.

[1180] 30. The compound according to clause 28 or 29, wherein a=2.

[1181] 31. A compound according to clause 28 or 29, wherein a=3.

[1182] 32. The compound according to clause 28, wherein b=3.

[1183] 33. A compound of formula (VIII*):

[1184]

[1185] 34. A compound of formula (IX*):

[1186]

[1187] 35. A compound according to clause 33 or 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.

[1188] 36. The compound according to clause 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro and 2'-deoxy.

[1189] 37. The compound according to any one of clauses 33 to 36, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[1190] 38. A compound according to clause 37, wherein the one or more degradation protection moieties are not present at the terminus 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 carrying the linker / ligand moiety.

[1191] 39. A compound according to clause 33, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 5' end of the second strand thereof.

[1192] 40. A compound according to clause 34, wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 adjacent phosphate groups at the 3' end of the second strand thereof.

[1193] 41. A process for preparing a compound according to any one of clauses 1 to 40, comprising reacting compounds of formula (X*) and (XI*):

[1194]

[1195] in,

[1196] r and s are independently integers from 1 to 16; and

[1197] Z is an oligonucleoside moiety;

[1198] And where appropriate, the ligand is deprotected and / or the second strand of the oligonucleotide is annealed.

[1199] 42. A process according to clause 41, for preparing a compound according to any one of clauses 6, 8-14, 16-33 and 35-40, wherein:

[1200] The compound of formula (X*) is of formula (Xa*):

[1201]

[1202] And the compound of formula (XI*) is formula (XIa*):

[1203]

[1204] wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 5' end of the second strand thereof.

[1205] 43. A process according to clause 41, for preparing a compound according to any one of clauses 5, 7, 9-13, 15-32 and 34-40, wherein:

[1206] The compound of formula (X*) is of formula (Xb*):

[1207]

[1208] And the compound of formula (XI*) is formula (XIa*):

[1209]

[1210] wherein the oligonucleoside comprises an RNA duplex comprising a first and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, and the second strand is at least partially complementary to the first strand, 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 the second strand thereof.

[1211] 44. A method according to clause 42 or 43, wherein:

[1212] The compound of formula (XIa*) is formula (XIb*)

[1213]

[1214] 45. A compound of formula (X*)

[1215]

[1216] in:

[1217] r is independently an integer from 0 to 16; and

[1218] Z is an oligonucleoside moiety.

[1219] 46. ​​A compound having the formula (Xa*)

[1220]

[1221] 47. A compound having the formula (Xb*)

[1222]

[1223] 48. A compound having the formula (XI*)

[1224]

[1225] in,

[1226] s is independently an integer from 0 to 16; and

[1227] Z is an oligonucleoside moiety.

[1228] 49. A compound having the formula (XIa*)

[1229]

[1230] 50. A compound having the formula (XIb*)

[1231]

[1232] 51. Use of a compound according to clause 45 and any one of clauses 48 to 50 for the preparation of a compound according to any one of clauses 1 to 40.

[1233] 52. Use of a compound according to clause 46 for preparing a compound according to any one of clauses 6, 8-14, 16-33, and 35-40.

[1234] 53. Use of a compound according to clause 47 for preparing a compound according to any one of clauses 5, 7, 9-13, 15-32, and 34-40.

[1235] 54. A compound or composition obtained or obtainable by a method according to any one of clauses 41-44.

[1236] 55. A pharmaceutical composition comprising a compound according to any one of clauses 1 to 40, and a pharmaceutically acceptable carrier, diluent or excipient.

[1237] A compound according to any one of clauses 1 to 40, for use in therapy.

[1238] Example

[1239] The present application will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present application. It should be understood that the embodiments and implementations described herein are only for illustrative purposes, and various modifications or changes will be suggested to those skilled in the art according to them, and will be included in the spirit and scope of the present application and the scope of the appended claims.

[1240] Example 1—Target Identification

[1241] background

[1242] All biological functions, including coagulation (physiological control of bleeding), arise from the coordinated interplay of hundreds of interacting molecules, primarily proteins, and can be considered as emergent functional outcomes of protein-protein interaction networks, where each node in the network is a protein and each edge connecting proteins can represent a range of possible interaction types, from complex formation to catalytic activation.

[1243] Historically, these functions have been represented as simple linear pathways. However, as knowledge has grown, it has become clear that pathways are much more complex and that the lowest level of complexity that can adequately represent the functional properties of a biological process and capture the characteristics of resilience to perturbations and robustness to random damage to individual components is a network.

[1244] Networks that perform biological functions may consist of several interacting canonical pathways as well as additional proteins that are primarily engaged when canonical functions are perturbed. Therefore, it is critical to develop methods that can model this complexity in a meaningful and tractable way and generate targeted hypotheses that take into account the inherent resistance to change due to network robustness. Figure 8 shows an example of the additional complexity captured by this framework.

[1245] The oversimplification of biology and the failure to make rational drug target selection based on realistic models of biological processes have resulted in low drug discovery success rates. Furthermore, there has been a lack of rigorous, objective methods for making network models of processes and for prioritizing protein targets within these models. As a result, target selection decisions are often made ad hoc based on bias or evidence that is unrelated to functional models.

[1246] Coagulation is a complex process involving many cell types and proteins and is tightly regulated by a system of pro- and anticoagulant mechanisms. In hemophilia and other bleeding disorders, genetic failures of individual protein components have varying degrees of impact on the ability of the coagulation system to control bleeding. There is an unmet need for well-tolerated, safe treatments that improve coagulation and are easy for patients to comply with.

[1247] The process of identifying processes and goals

[1248] The inventors used a network biology approach to create a network model of blood coagulation ( Fig. 9 ). The network model aims to capture all the important proteins involved in the process, their connections and, importantly, the direction of information flow between pairs of proteins.

[1249] The inventors 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 for information to the rest of the network, an influencer of key proteins, and the extent to which influencers are influenced by or influence other proteins (based on the absolute and relative numbers and directions of inputs and outputs). Directional information also allows for estimation of hierarchical relationships between proteins. Proteins that are higher in the hierarchy and have certain properties may be more preferred than other proteins with similar properties. The relative specificity and extent of each property relative to other properties allows the inventors to score and rank proteins according to their targeting suitability.

[1250] The ability to characterize these targets based on network relationships enables judgments to be made about selectivity and magnitude of impact in a chosen context, thereby determining the suitability of each target for a specific indication.

[1251] This allowed the inventors to identify targets that could improve bleeding control in hemophilia and other bleeding disorders. The assays used for this purpose were specifically designed to search for novel and non-obvious indirect enhancers of bleeding that, after knockdown in hepatocytes by GalNAc-siRNA, could be beneficial in hemophilia by inhibiting the production of secretory proteins involved in coagulation regulation.

[1252] In particular, a selected set of coagulation-related pathway proteomes was used to algorithmically generate a set of directed network models using an internal proprietary database of protein-protein relationships that includes the "directions" of interactions. This directional information comes from a range of public and internal databases, supplemented by estimated directions from natural language processing of scientific publication text. The inventors' network construction algorithms use these information sources to build "directed" models of key biological processes.

[1253] Proprietary analytical techniques are then applied to the network model to identify pharmacologically actionable targets from the network whose knockdown will have a significant impact on the network and, in turn, on the biological function being modeled. These algorithms make extensive use of directional information and hierarchical relationships to identify targets with a set of specific properties that make them good siRNA targets. Targets are then further filtered by protein class and hepatocyte-specific properties based on therapeutic requirements.

[1254] The workflow described above, using proprietary data resources and network node metrics, identified two clinically validated coagulation modulators that are being developed for the treatment of bleeding disorders (one of which is AT3, shown above). In addition, it highlighted a novel, less obvious coagulation modulator, protein Z-dependent protease inhibitor (ZPI), which our analysis predicted was superior to either on a metric estimating indirect effects and therefore may have a superior safety profile ( Fig. 9 ).

[1255] Example 2: Synthesis of Tether 1

[1256] General experimental conditions:

[1257] Thin layer chromatography (TLC) was performed on aluminum plates coated with silica with a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were visualized under UV light (254 nm); or according to Stahl (from Sigma-Aldrich), they were visualized by spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent followed by heating. A Biotage Isola One flash chromatograph equipped with a dual variable UV wavelength detector (200-400 nm) was used, using a Biotage Flash chromatography was performed using Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1258] All moisture-sensitive reactions were performed under anhydrous conditions using dry glassware, anhydrous solvents, and argon atmosphere. All commercial reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH+Co. KG. D-galactosamine pentaacetate was purchased from AK scientific.

[1259] HPLC / ESI-MS was performed using a Dionex UltiMate 3000RS UHPLC system and a ThermoScientific MSQ Plus mass spectrometer. The chromatographic column was an Acquity UPLC Protein BEH C4 column ( 1.7 μm, 2.1 x 100 mm), column temperature was 60°C. Solvent system consisted of solvent A in H2O containing 0.1% formic acid, and solvent B in acetonitrile (ACN) containing 0.1% formic acid. A gradient of 5-100% B in 15 minutes at a flow rate of 0.4 mL / min was used. Detector and conditions: Corona supercharged aerosol detection (from the European Space Agency (esa)). Nebulizer temperature: 25°C. N2 pressure: 35.1 psi. Filter: Corona.

[1260] 1 H and 13 C NMR spectra were acquired at room temperature on a Varian spectrometer at 500 MHz (1H NMR) and 125 MHz ( 13 C NMR) records. Chemical shifts are in ppm and are referenced to the residual solvent peak (CDCl 3-1 H NMR: δ at 7.26 ppm, 13 CNMR δ is 77.2ppm; DMSO-d6-1HNMR: δ is 2.50ppm, 13 C NMR δ is 39.5 ppm). Coupling constants are in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m).

[1261] Synthetic route of coupling building block TriGalNAc_tether 1:

[1262]

[1263] Preparation of compound 2: D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reactant was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated NaHCO3 aqueous solution (100 mL) and water (100 ml). The organic layer was separated, dried over Na2SO4 and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM, 10 CV).

[1264] The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1265]

[1266] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq) and azide-PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq) were dissolved in anhydrous DCM (40 mL) under argon, and molecular sieves were added to the solution. (5g). The mixture was stirred at room temperature for 1h. TMSOTf (0.77g, 3.49mmol, 0.5eq.) was then added to the mixture and the reaction was stirred overnight. The molecular sieves were filtered, and the filtrate was diluted with DCM (100mL), washed with cold saturated NaHCO3 aqueous solution (100mL) and water (100ml). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM, 10CV) to give the title product as a light yellow oil (3.10g, 88%, rf=0.25 (2% MeOH in DCM)). MS: calculated value is C 20 H 32 N4O 11 504.21. Found 505.4. 1H 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 (125MHz, CDCl3) δ170.6(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).

[1267]

[1268] Preparation of compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1: 1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed using vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through diatomaceous earth 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 can be used without further purification. MS: calculated value is C 20 H 34 N2O 11 The measured value is 479.4.

[1269]

[1270] Preparation of compound 7:

[1271] 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 Na was added while stirring vigorously CO (0.18 g, 1.7 mmol, 0.25 eq.). 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 CH Cl (100 mL), and washed with water (100 ml). The organic layer was separated and dried with Na SO. The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane, 12 CV) to afford the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane). MS: calculated for C 33 H 53 NO 11 639.3. Found 640.9. 1H NMR (500MHz, 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). 13C NMR (125MHz, DMSO-d6) δ170.3(3xC), 154.5(C), 137.1(C), 128.2(2xCH), 127.7(CH), 127.6(2x CH), 79.7(3xC), 68.4(3xCH2), 66.8(3xCH2), 64.9(C), 58.7(CH2), 35.8(3xCH2), 27.7(9xCH3).

[1272]

[1273] Preparation of compound 8: Cbz-NH-tris-Boc ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) 3 times and dried under high vacuum to obtain the compound in the form of TFA salt (0.183 g, 98%). The compound was used without further purification. MS: calculated value was C 21 H 29 The NO value is 471.6. The measured value is 472.4.

[1274]

[1275] 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-benzotriazole-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, and the residue was dissolved in DCM (100 mL) and washed with saturated aqueous NaHCO3 (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 14CV). The product was obtained as a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: calculated value is C 81 H 125 N7O 41of 1852.9. The measured value is 1854.7. 1H 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 (125MHz, DMSO-d6) δ170.1(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).

[1276]

[1277] Preparation of compound 10: The trident 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 using vacuum / argon cycles (3x) and hydrogenated overnight under balloon pressure. After the reaction was completed, 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 in the next step without further purification. A light yellow oily product (0.24 g, quantitative yield) was obtained. MS: The calculated value of C 73 H 119 N7O 39 The measured value is 1718.8. The actual value is 1719.3.

[1278]

[1279] Preparation of compound 11: Commercially available bis(N-hydroxysuccinimide) suberic acid (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 the solution. The reactants were stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 16 CV). A white solid product (1.54 g, 43%, rf=0.71 (5% MeOH in DCM)) was obtained. MS: calculated value is C 15 H 23 N5O5: 353.4.

[1280] The actual measured value is 354.3.

[1281]

[1282] Preparation of TriGalNAc (12): Trigalnac 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) under argon, and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reactants were stirred at room temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc (100 mL) and washed with water (100 ml). The organic layer was separated and dried over Na2SO4. The solvent was evaporated and the crude material was purified by flash chromatography (elution gradient: 0-10% MeOH in DCM, 20CV) to give the title compound as a white fluffy solid (0.27 g, 67%, rf=0.5 (10% MeOH in DCM)). MS: calculated value is C 84 H 137 N 11 O4's 1957.1. The measured value is 1959.6.

[1283] Coupling of tether 1 to siRNA strand: 5'- or 3'-terminal monofluorocyclooctyne (MFCO) coupling

[1284] 5' end MFCO conjugation

[1285]

[1286] 3' end MFCO coupling

[1287]

[1288] General conditions for MFCO coupling: The amine-modified single strand was dissolved in 50 mM carbonate / bicarbonate buffer pH 9.6 / dimethyl sulfoxide (DMSO) 4:6 (v / v) at a concentration of 700 OD / mL, and one molar equivalent of a 35 mM solution of MFCO-C6-NHS ester (Berry & Associates, Cat. # LK 4300) in DMF was added to the solution. The reaction was carried out at room temperature and another molar equivalent of MFCO solution was added after 1 h. The reaction was allowed to proceed for another hour and monitored by LC / MS. At least two molar equivalents of excess MFCO NHS ester reagent relative to the amino-modified oligonucleotide were required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered through a 1.2 μm filter from Sartorius, and then incubated at 4 °C for 1 hr. Purification was by reverse phase (RP HPLC) on a Pure instrument (GE Healthcare).

[1289] Purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM TEAAc pH 7 and buffer B was 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60°C. UV traces were recorded at 280 nm. A gradient of 0-100% B was applied over 60 column volumes.

[1290] Fractions containing full-length conjugated oligonucleotides were pooled, precipitated in a refrigerator with 3M NaOAc, pH 5.2, and 85% ethanol, and the collected precipitates were dissolved in water. Samples were desalted by size exclusion chromatography and concentrated using a speed vacuum concentrator to afford conjugated oligonucleotides in 40-80% isolated yields.

[1291] 5'-GalNAc-T1 conjugate

[1292]

[1293] 3'-GalNAc-T1 conjugate

[1294]

[1295] General procedure for TriGalNAc coupling: MFCO-modified single strands were dissolved in water at a concentration of 2000 OD / mL, and an equivalent solution of compound 12 (10 mM) in DMF was added to the solution. The reaction was carried out at room temperature, and 0.7 molar equivalents of compound 12 solution were added after 3 h. The reaction was allowed to proceed overnight and the completion was monitored by LCMS. The conjugate was diluted 15-fold in water, filtered through a 1.2 μm filter from Sartorius, and then Purification was performed by RP HPLC on a Pure instrument (GE Healthcare).

[1296] RP HPLC purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM triethylammonium acetate pH 7 and buffer B was 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60°C. UV traces were recorded at 280 nm. A gradient of 0-100% B was used in 60 column volumes.

[1297] The components containing the full-length coupled oligonucleotides were combined, precipitated in a refrigerator with 3M NaOAc, pH 5.2 and 85% ethanol, and the collected precipitates were dissolved in water to obtain an oligonucleotide solution of about 1000 OD / mL. O-acetate was removed by adding 20% ​​ammonia. The quantitative removal of these protecting groups was verified by LC-MS.

[1298] exist The conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on a Pure (GE Healthcare) instrument to obtain the conjugated oligonucleotides in isolated yields of 50-70%.

[1299] The following scheme further illustrates the synthetic route:

[1300] Scenario 1:

[1301]

[1302] Scenario 2:

[1303]

[1304] Solution 3:

[1305] 5' end MFCO coupling

[1306]

[1307] 3' end MFCO coupling

[1308]

[1309] Solution 4:

[1310]

[1311] Solution 5:

[1312]

[1313] Example 3: Double-strand annealing

[1314] To generate the desired siRNA duplex, the two complementary strands were annealed by mixing 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 within 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[1315] The HPLC was carried out on a Dionex Ultimate 3000 (Thermo Fisher Scientific) system by Superdex TM Analytical SEC HPLC analysis of duplexes on a 75Increase 5 / 150GL column 5x 153-158mm (Cytiva). The mobile phase consisted of 1x PBS containing 10% acetonitrile. An isocratic gradient was run at a flow rate of 1.5mL / min over 10 minutes at room temperature. UV traces at 260 and 280nm were recorded. 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).

[1316] Example 4: Synthesis of Tether 2

[1317] General experimental conditions:

[1318] Thin layer chromatography (TLC) was performed on silica-coated aluminum plates with a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were visualized under UV light (254 nm); or according to Stahl (from Sigma-Aldrich), they were visualized by spraying with 5% H2SO4 in methanol (MeOH) or ninhydrin reagent followed by heating. A Biotage Isola One flash chromatograph equipped with a dual variable UV wavelength detector (200-400 nm) was used, using a Biotage Flash chromatography was performed using Silica 10, 25, 50 or 100 g columns (Uppsala, Sweden).

[1319] All moisture-sensitive reactions were performed under anhydrous conditions, using dry glassware, anhydrous solvents, and under an argon atmosphere. All commercial reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH+Co.KG. D-galactosamine pentaacetate was purchased from AK Technology.

[1320] HPLC / ESI-MS was performed using a Dionex UltiMate 3000RS UHPLC system and a ThermoScientific MSQ Plus mass spectrometer. The chromatographic column was an Acquity UPLC Protein BEH C4 column ( 1.7 μm, 2.1 x 100 mm), column temperature was 60°C. Solvent system consisted of solvent A in H2O containing 0.1% formic acid, and solvent B in acetonitrile (ACN) containing 0.1% formic acid. A gradient of 5-100% B in 15 minutes at a flow rate of 0.4 mL / min was used. Detector and conditions: Corona supercharged aerosol detection (from the European Space Agency (esa)). Nebulizer temperature: 25°C. N2 pressure: 35.1 psi. Filter: Corona.

[1321] 1 H and 13 C NMR spectra were acquired at room temperature on a Varian spectrometer at 500 MHz (1H NMR) and 125 MHz ( 13 C NMR) records. Chemical shifts are in ppm and are referenced to the residual solvent peak (CDCl 3-1 H NMR: δ at 7.26 ppm, 13 CNMR δ is 77.2ppm; DMSO-d6-1HNMR: δ is 2.50ppm, 13 C NMR δ is 39.5 ppm). Coupling constants are in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t) or multiplet (m).

[1322] Synthetic route of coupling building block TriGalNAc_tether 2:

[1323]

[1324] Preparation of compound 2: D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reactant was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated NaHCO3 aqueous solution (100 mL) and water (100 ml). The organic layer was separated, dried over Na2SO4 and concentrated to give the title compound as a yellow oil, which was purified by flash chromatography (gradient elution: 0-10% MeOH in DCM, 10 CV).

[1325] The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[1326]

[1327] Preparation of compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq) and azide-PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq) were dissolved in anhydrous DCM (40 mL) under argon, and molecular sieves were added to the solution. (5g). The mixture was stirred at room temperature for 1h. TMSOTf (0.77g, 3.49mmol, 0.5eq.) was then added to the mixture and the reaction was stirred overnight. The molecular sieves were filtered, and the filtrate was diluted with DCM (100mL), washed with cold saturated NaHCO3 aqueous solution (100mL) and water (100ml). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0-3% MeOH in DCM, 10CV) to give the title product as a light yellow oil (3.10g, 88%, rf=0.25 (2% MeOH in DCM)). MS: calculated value is C 20 H 32 N4O 11 504.21. Found 505.4. 1H 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 (125MHz, CDCl3) δ170.6(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).

[1328]

[1329] Preparation of compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1: 1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed using vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through diatomaceous earth 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 can be used without further purification. MS: calculated value is C 20 H 34 N2O 11 The measured value is 479.4.

[1330]

[1331] Preparation of compound 7:

[1332] 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 Na was added while stirring vigorously CO (0.18 g, 1.7 mmol, 0.25 eq.). 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 CH Cl (100 mL), and washed with water (100 ml). The organic layer was separated and dried with Na SO. The solvent was removed under reduced pressure and the resulting crude material was purified by flash chromatography (gradient elution: 0-10% EtOAc in cyclohexane, 12 CV) to afford the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane). MS: calculated for C 33 H 53 NO11 639.3. Found 640.9. 1H NMR (500MHz, DMSO-d6) δ 7.38-7.26 (m, 5H), 4.97 (s, 2H), 3.54 (t, 6H), 3.50 (s, 6H), 2.38 (t, 6H), 1.39 (s, 27H). 13 C NMR (125MHz, DMSO-d6) δ170.3(3xC), 154.5(C), 137.1(C), 128.2(2xCH), 127.7(CH), 127.6(2x CH), 79.7(3xC), 68.4(3xCH2), 66.8(3xCH2), 64.9(C), 58.7(CH2), 35.8(3xCH2), 27.7(9xCH3).

[1333]

[1334] Preparation of compound 8: Cbz-NH-tris-Boc ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) 3 times and dried under high vacuum to obtain the compound in the form of TFA salt (0.183 g, 98%). The compound was used without further purification. MS: calculated value was C 21 H 29 The NO value is 471.6. The measured value is 472.4.

[1335]

[1336] 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-benzotriazole-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, and the residue was dissolved in DCM (100 mL) and washed with saturated aqueous NaHCO3 (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 14CV). The product was obtained as a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: calculated value is C 81 H 125 N7O 41 of 1852.9. The measured value is 1854.7. 1HNMR (500MHz, DMSO-d6) δ7.90-7.80 (m, 10H), 7.65-7.62 (m, 4H), 7.47-7.43 (m, 3H), 7.38-7.32 (m, 8H), 5.24-5.22 (m, 3H), 5.02-4.97 (m, 4H), 4.60-4.57 (m, 3H), 4.07-3.90 (m 10H), 3.67-3.36 (m, 70H), 3.23-3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80-1.78 (m, 17H). 13C NMR (125MHz, DMSO-d6) δ170.1(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).

[1337]

[1338] Preparation of compound 10: The trident 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 using vacuum / argon cycles (3x) and hydrogenated overnight under balloon pressure. After the reaction was completed, 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 in the next step without further purification. A light yellow oily product (0.24 g, quantitative yield) was obtained. MS: calculated value is C 73 H 119 N7O 39 The measured value is 1718.8. The actual value is 1719.3.

[1339]

[1340] Preparation of compound 14: Tridentate 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) under argon. A solution of compound 13 (0.14 g, 0.53 mmol, 2.0 eq.) in DCM (5 mL) was added dropwise to the mixture. The reaction was stirred at room temperature overnight. The solvent was removed and the residue was dissolved in EtOAc (50 mL), washed with water (50 mL) and dried over Na2SO4. The solvent was evaporated and the crude material was purified by flash chromatography (gradient elution: 0-5% MeOH in DCM, 20CV). A white fluffy solid product (0.25 g, 48%, rf=0.4 (10% MeOH in DCM)) was obtained. MS: calculated value is C88 H 137 N7O 42 The measured value is 1965.1. The actual value is 1965.6.

[1341]

[1342] Preparation of TriGalNAc (15): Trigalnac compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) was dissolved in EtOAc (15 mL) and Pd / C (40 mg) was added. The reaction mixture was degassed using vacuum / argon cycles (3x) and hydrogenated overnight under balloon pressure. The completion of the reaction was monitored by mass spectrometry and the resulting mixture was filtered through a thin pad of diatomaceous earth. The solvent was removed under reduced pressure and the resulting residue was dried overnight under high vacuum. The residue was used for coupling with oligonucleosides without further purification (0.28 g, quantitative yield). MS: calculated value is C 81 H 131 N7O 42 The measured value is 1875.3.

[1343] Coupling of tether 2 to siRNA strand: Coupling of TriGalNAc tether 2 (GalNAc-T2) at the 5'-end or 3'-end

[1344] 5'-GalNAc-T2 conjugate

[1345]

[1346] 3'-GalNAc-T2 conjugate

[1347]

[1348] Preparation of TriGalNAc tether 2 NHS ester: To a solution of carboxylic acid tether 2 (compound 15, 227 mg, 121 μmol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol) were added. The solution was stirred at room temperature for 18 hours and used in the subsequent coupling reaction without purification.

[1349] General procedure for coupling of TriGalNAc tether 2: The amine-modified single strand was dissolved in 50 mM carbonate / bicarbonate buffer pH 9.6 / DMSO 4:6 (v / v) at a concentration of 700 OD / mL, and one molar equivalent of tether 2 NHS ester (57 mM) solution in DMF was added to the solution. The reaction was carried out at room temperature, and another molar equivalent of NHS ester solution was added after 1 h. The reaction was carried out 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 oligonucleoside are required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15 times with water, filtered once through a 1.2 μm filter from Sartorius, and then in 5% PBS. Purification was performed by reverse phase (RP HPLC) on a Pure (GE Healthcare) instrument.

[1350] Purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM TEAA pH 7 and buffer B was 95% acetonitrile in buffer A. The flow rate was 10 mL / min and the temperature was 60°C. UV traces were recorded at 280 nm. A gradient of 0-100% B was applied over 60 column volumes.

[1351] Fractions containing full-length coupled oligonucleotides were combined, precipitated in a refrigerator with 3M NaOAc, pH 5.2 and 85% ethanol, and then dissolved in water at 1000 OD / mL. O-acetate was removed with 20% aqueous ammonium hydroxide until complete (monitored by LC-MS).

[1352] exist The conjugates were desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on a Pure (GE Healthcare) instrument to generate the conjugated oligonucleotides with isolated yields of 60-80%.

[1353] The conjugates were characterized by HPLC-MS analysis on a Dionex Ultimate3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics) using a 2.1 x 50 mm XBridge C18 column (Waters). Buffer A was 16.3 mM triethylamine, 100 mM HFIP in 1% MeOH aqueous solution, and buffer B was 95% MeOH in buffer A. A flow rate of 250 μL / min and a temperature of 60° C. were used. UV traces at 260 and 280 nm were recorded. A gradient of 1-100% B was used in 31 minutes.

[1354] The following scheme further illustrates the synthetic route:

[1355] Solution 6:

[1356]

[1357] Solution 7:

[1358]

[1359] Solution 8:

[1360]

[1361] Solution 9:

[1362]

[1363] Example 5: Double-strand annealing

[1364] To generate the desired siRNA duplex, the two complementary strands were annealed by mixing 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 within 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[1365] The HPLC was carried out on a Dionex Ultimate 3000 (Thermo Fisher Scientific) system by Superdex TM Analytical SEC HPLC analysis of duplexes on a 75Increase 5 / 150GL column 5x 153-158mm (Cytiva). The mobile phase consisted of 1x PBS containing 10% acetonitrile. An isocratic gradient was run at a flow rate of 1.5mL / min over 10 minutes at room temperature. UV traces at 260 and 280nm were recorded. 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).

[1366] Example 6: Alternative synthetic route for coupling building block TRIGALNAC_tether 2:

[1367]

[1368]

[1369] Coupling of tether 2 to siRNA strand: Coupling of TriGalNAc tether 2 (GalNAc-T2) at the 5'-end or 3'-end

[1370] Coupling conditions

[1371]

[1372] Pre-activation: To a solution of compound 15 (16 umol, 4 eq.) in DMF (160 μL) at 25°C, TFA-O-PFP (15 μL, 21 eq.) was added, followed by DIPEA (23 μL, 32 eq.). The tube was shaken at 25°C for 2 h. The reaction was quenched with H2O (10 μL).

[1373] Coupling: The resulting mixture was diluted with DMF (400 μl) and then an oligoamine solution (4.0 μmol in 10x PBS, pH 7.4, 500 μl; final oligomer concentration in organic and aqueous solutions: 4 μmol / ml = 4 mM) was added. The tube was shaken at 25°C for 16 h and the reaction was analyzed by LCMS. The resulting mixture was treated with 28% NH4OH (4.5 ml) and shaken at 25°C for 2 h. The mixture was analyzed by LCMS, concentrated, and purified by IP-RP HPLC to produce an oligonucleotide coupled to a tethered 2-GalNAc.

[1374] 5'-GalNAc-T2 conjugate

[1375]

[1376] 3'-GalNAc-T2 conjugate

[1377]

[1378] Example 7: Solid Phase Synthesis: Scale ≤ 1 μM OL

[1379] Synthesis of siRNA sense and antisense strands was performed on a MerMade 192X 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).

[1380] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[1381] The 2'-O-methylphosphoramidites used were as follows: 5'-(4,4'-dimethoxytrityl)-N-benzoyladenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetylcytidine 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.

[1382] The 2'-F phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1383] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except 2'-O-methyl-uridine phosphoramidite which was dissolved in DMF / MeCN (1:4, v / v). Iodine was used as an oxidant at 0.02 M in acetonitrile / pyridine / H2O (DNAchem). Thiolation of phosphorothioate bonds was performed with 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v. 5-Ethylmercaptotetrazole (ETT) was used as an activator solution at 0.25 M mM in acetonitrile.

[1384] The reverse abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[1385] In each cycle, DMT was removed by 3% TCA demodulation solution in DCM (DNAchem).

[1386] The coupling time was 180 seconds. The oxidant contact time was set to 80 seconds and the thiolation time was 2*100 seconds.

[1387] At the end of the synthesis, the oligonucleotides were cleaved from the solid support using a 4:1 (v / v) NH4OH:EtOH solution at 45°C (TCI) for 20 h. The solid support was then filtered off, the filter washed thoroughly with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[1388] Oligonucleotides were treated to form sodium salts by ultracentrifugation using an Amicon Ultra-2 centrifugal filter device, PBS buffer (10x, Teknova, pH 7.4, sterile) or by EtOH precipitation from 1 M sodium acetate.

[1389] Single-strand identity was assessed by MS ESI-PCR followed by annealing in water to form the final double-stranded siRNA and assessment of double-strand purity by size exclusion chromatography.

[1390] Example 8: Solid Phase Synthesis: Scale ≥ 5 μM OL

[1391] The synthesis of siRNA sense and antisense strands was performed on a MerMade 12 synthesizer at a scale of 5 μMOL with 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). On the solid support, the sense strand for 3' coupling was synthesized at a concentration of 12 μmol.

[1392] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[1393] The 2'-O-methylphosphoramidites 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.

[1394] The 2'-F phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[1395] The reverse abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[1396] All phosphoramidites were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except 2'-O-methyl-uridine phosphoramidite which was dissolved in DMF / MeCN (1:4, v / v). Iodine was used as an oxidant at 0.02 M in acetonitrile / pyridine / H2O (DNAchem). Thiolation of phosphorothioate bonds was performed with 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 v / v. 5-Ethylmercaptotetrazole (ETT) was used as an activator solution at 0.25 M mM in acetonitrile.

[1397] In each cycle, DMT was removed by 3% TCA demodulation solution in DCM (DNAchem).

[1398] For chains synthesized on universal CPG, coupling was performed for 130 seconds with 8 equivalents of amidite. Oxidation time was 47 seconds and thiolation time was 210 seconds.

[1399] For the chains synthesized on 3'-PT-Amino-Modifier C6 CPG, couplings were performed 2*150 sec with 8 equivalents of phosphoramidite. Oxidation time was 47 sec and thiolation time was 250 sec.

[1400] At the end of the synthesis, the oligonucleotides were cleaved from the solid support using a 4:1 (v / v) NH4OH:EtOH solution at 45°C (TCI) for 20 h. The solid support was then filtered off, the filter washed thoroughly with H2O, and the volume of the combined solution was reduced by evaporation under reduced pressure.

[1401] Oligonucleotides were treated with EtOH precipitation using 1 M sodium acetate to form the sodium salt.

[1402] Single-stranded oligonucleotides were purified by Xbridge BEH C18 5 μm, IP-RPHPLC on a 19x150 mm (Waters) column, purification was performed using a gradient of increasing B in A. Mobile phase A: 240 mM HFIP, 7 mM TEA and 5% methanol in water; Mobile phase B: 240 mM HFIP, 7 mM TEA in methanol.

[1403] Single-chain purity and identity were assessed by UPLC / MS ESI on a Xbridge BEH C18 2.5 μm, 3x50 mm (Waters) column using an increasing gradient of B in A. Mobile phase A: 100 mM HFIP, 5 mM TEA in water; Mobile phase B: 20% Mobile phase A: 80% acetonitrile (v / v).

[1404] According to the scheme provided in any one of Examples 2, 4, and 6, the sense strand is coupled.

[1405] The sense and antisense strands were then annealed in water to form the final double-stranded siRNA, and the double-strand purity was assessed by size exclusion chromatography.

[1406] The scope of the present application is not limited to the specific embodiments disclosed, for example, these embodiments are provided to illustrate the various aspects of the present application. According to the description and teaching of this paper, various modifications to the composition and method will become apparent. Such changes can be implemented without departing from the true scope and spirit of the application, and these changes are intended to fall within the scope of the present application.

[1407] Example 9: In vivo efficacy data in a hemophilia mouse model

[1408] A total of 20 hemophilia A mice (Bi, L., Lawler, A., Antonarakis, S. et al. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 10, 119–121 (1995). https: / / doi.org / 10.1038 / ng0595-119) and 10 WT mice were used in this study.

[1409]

[1410] Eight days before induction of knee joint bleeding, mice were injected subcutaneously (sc) with the GalNAc-siRNA construct ETXM1184 or vehicle (0.9% saline) at a dose volume of 5 ml / kg.

[1411] To induce knee bleeding, mice were weighed and anesthetized using isoflurane inhalation anesthetic. Both legs were shaved to expose the knee joints. Mice were subcutaneously injected with buprenorphine 10 ml / kg for analgesia, and the diameter of both knees was measured with an electronic caliper. Subsequently, both knees were wiped with 70% ethanol.

[1412] A 30G sterile hypodermic needle was inserted into the infrapatellar ligament of one knee. The injected knee was randomized between left and right, and the side of the injection was recorded. The mouse was removed from anesthesia and allowed to recover in a heated cage before being returned to its original cage.

[1413] Mice were monitored regularly during the first 6 h and buprenorphine (10 ml / kg) was injected subcutaneously 6 h after injury for analgesia. Visual bleeding scores (VBS) of the injured knees were assessed at 72 h and 10 days after injury.

[1414] All mice were carefully examined daily for clinical signs of excessive blood loss. Mice showing clinical signs of excessive blood loss, piloerection, avoidance of companions, or baring of teeth were killed for euthanasia.

[1415] Mice were withdrawn from the study 10 days after injury.

[1416] Under isoflurane anesthesia, citrate anticoagulated blood samples were collected by cardiac puncture, and plasma was prepared and aliquoted, frozen on dry ice and stored at -80°C. To this end, blood was collected in a 1:9 ratio into 3.8% sodium citrate and then centrifuged at 7000 x g for 10 minutes at 4°C. Specifically, the following steps were performed:

[1417] 1. Blood collection by cardiac puncture.

[1418] 2. Rinse the syringe and needle with sodium citrate solution (3.8%), leaving the solution in the center of the syringe (~30 μl).

[1419] 3. After blood collection, drain the sample into a 1.5ml microcentrifuge tube and make sure to add enough sodium citrate solution (3.8%) to achieve a 1:9 ratio of sodium citrate to blood. Add the sodium citrate solution to the side of the tube, not directly to the sample. Invert 4-6 times to mix. If the sample is not centrifuged immediately, store it in a refrigerator (if available), or wrap the collection tube in ice and continue to invert the collection tube regularly.

[1420] 4. Centrifuge the sample as quickly as possible at 7000 x g for 10 minutes at 4°C.

[1421] 5. Remove all plasma from the sample and place into a fresh microcentrifuge tube.

[1422] 6. Aliquot the plasma into pre-labeled tubes (Thermo Scientific; 10775974) as follows:

[1423] 30 μl for potential TGA analysis

[1424] 100 μl for potential APTT analysis

[1425] All remaining fractions were used for potential target protein abundance analysis.

[1426] 7. Immediately place all aliquots on wet / dry ice.

[1427] 8. Transport samples on wet / dry ice.

[1428] 9. Transfer the samples to a -20℃ / -80℃ freezer and store until use.

[1429] The liver was removed and up to 3 parts of each liver lobe were subsequently placed in RNA and stored at 4°C for 24-72 hours. The tissue was then blotted dry, weighed and stored at -80°C. Specifically, the following steps were performed:

[1430] 1. Immediately after cardiac puncture, kill the mice by cervical dislocation.

[1431] 2. Make an incision in the abdominal wall and remove the liver as quickly as possible.

[1432] 3. Place the liver on a petri dish on wet ice to minimize sample degradation.

[1433] 4. Cut 3 ~50 mg liver pieces from each of the following lobes: left lateral lobe, median lobe, right lateral lobe, and caudate lobe. Immediately place these liver pieces into pre-labeled tubes (1.5 ml microcentrifuge tubes) containing 500 μl RNAlater and place the collection tubes on wet ice.

[1434] a. Ship on wet ice and store at 4°C.

[1435] b. After 24-72 hours, wipe the liver sample dry and weigh it. Record the weight on the terminal form.

[1436] c. Transfer to -80°C for long-term storage.

[1437] 5. Collect any excess liver into separate pre-labeled collection tubes (2 ml microcentrifuge tubes).

[1438] a. Freeze on dry ice for future analysis.

[1439] b. Transport samples on dry ice.

[1440] c. Transfer to -80°C for long-term storage.

[1441] 6. Clean all dissection tools between handling different animals to prevent any cross contamination.

[1442] The skin was removed from the legs and the knee joints were measured. The legs were then placed in 10% formalin followed by decalcification and section preparation. Specifically, the following steps were performed:

[1443] 1. After removal of the liver, measure and record the diameters of the injured and uninjured knee joints.

[1444] 2. Remove the skin from both knees. Perform visual bleeding scores and measure the knee joints.

[1445] 3. Dissect the leg from the top of the femur to the ankle joint, removing some excess muscle, taking care not to cause any damage to the knee and associated structures. Place the knee in a pre-labeled tube (7 ml compact tube) containing 10% neutral buffered formalin for histological analysis.

[1446] On days 3 and 10 after induction of knee joint bleeding, visual bleeding scores were significantly reduced in hemophilia A mice that received the GalNAc-siRNA construct ETXM1184 compared with those in hemophilia A mice that received the vehicle (0.9% saline) (see Fig. 10A In addition, the knee diameter of mice that received the GalNAc-siRNA construct ETXM1184 recovered faster after induction of knee bleeding compared with mice that received the vehicle ( Fig.11A This observation was confirmed by comparing the difference in diameter between injured and non-injured skinned knee joints ( Fig. 11B ).

[1447] Analysis of hemophilia A mice 10 days after injury further showed that hemophilia A mice that received the GalNAc-siRNA construct ETXM1184 had less myeloproliferation ( Fig. 12A ), milder osteoarthritis ( Fig. 12B ), milder chondrocyte degeneration / necrosis ( Fig. 12C ), mild bleeding ( Fig.12D ), milder hemosiderin deposition ( Fig.12E ), milder hematoma ( Fig.12F ), milder osteoclastogenic bone resorption ( Figure 12G ), milder osteolysis ( Fig.12H), mild periostitis ( Fig.12I ), mild subchondral bone sclerosis ( Fig.12J ), milder tendinosis ( Figure 12K ), milder tendinitis ( Figure 12L ), milder tenosynovitis ( Figure 12M ).

Claims

1. An inhibitor of ZPI expression and / or function for use in preventing or treating diseases associated with coagulation disorders such as hemophilia.

2. An inhibitor of ZPI expression and / or function according to claim 1, wherein the inhibitor is coupled to one or more ligand moieties.

3. An inhibitor of ZPI expression and / or function according to claim 1 or 2, wherein the inhibitor is a siRNA oligomer.

4. An inhibitor of ZPI expression and / or function, wherein the inhibitor is coupled to one or more ligand moieties. The inhibitor according to claim 4 , wherein the inhibitor is a siRNA oligomer.

6. An inhibitor of ZPI expression and / or function, wherein the inhibitor is a siRNA oligomer.

7. The inhibitor of claim 6, wherein the inhibitor comprises a siRNA oligomer coupled to one or more ligand moieties.

8. The inhibitor according to claim 2, 4, 5 or 7, or the inhibitor for use therein, wherein the one or more ligand moieties comprise one or more GalNAc ligands or comprise one or more GalNAc ligand derivatives.

9. The inhibitor according to claim 2, 4, 5 or 7, or an inhibitor for use therein, wherein the one or more ligand moieties comprise one or more GalNAc ligand derivatives.

10. The inhibitor or the inhibitor for use according to one or more of the preceding claims, wherein the target of the inhibitor is ZPI.

11. The inhibitor according to one or more of the preceding claims, or the inhibitor for use therein, which is a siRNA oligomer having a first strand and a second strand, wherein: i) the length of the first strand of the siRNA is 15-30 nucleosides, preferably 19-25 nucleosides, more preferably 23 or 25 nucleosides; even more preferably 23 nucleosides; and / or ii) The length of the second strand of the siRNA is 15-30 nucleosides, preferably 19-25 nucleosides, and more preferably 21 nucleosides.

12. The inhibitor according to claim 11 or the inhibitor for use, wherein the second sense strand further comprises one or more abasic nucleosides in the terminal region of the second strand, and wherein the abasic nucleosides are linked to adjacent nucleosides via reverse internucleoside bonds.

13. The inhibitor according to claim 12 or the inhibitor for use thereof, wherein the second chain comprises: i 2 or more abasic nucleosides in the terminal region of the second strand; and / or ii. 2 or more abasic nucleosides in the 5' or 3' terminal region of the second strand; and / or iii. 2 or more abasic nucleosides at the 5' or 3' terminal region of the second strand, wherein the abasic nucleosides are present in a pendant form as described herein; and / or iv. 2 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 v 2 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 vi. in the terminal region of the second strand, at least one abasic nucleoside is linked to an adjacent abasic nucleoside via an inverted internucleoside linkage; and / or vii. at least one abasic nucleoside is linked to an adjacent abasic nucleoside via a reverse internucleoside linkage in the 5' or 3' region of the second strand; and / or viii an abasic nucleoside as the penultimate nucleoside, which is linked to a nucleoside as a non-terminal nucleoside (referred to herein as the penultimate nucleoside) via a reverse bond; and / or ix an abasic nucleoside that is the two terminal nucleosides joined via a 5'-3' bond when the strand is read in a direction toward the end; x is an abasic nucleoside that is the 2 terminal nucleosides connected via a 3'-5' bond when the strand is read in the terminal direction including the terminal nucleoside; xi as an abasic nucleoside at the terminal 2 positions, wherein the penultimate nucleoside is linked to the penultimate nucleoside via the reverse bond, and wherein the reverse bond is a 5-5' reverse bond or a 3'-3' reverse bond; xii as an abasic nucleoside at the terminal 2 positions, wherein the penultimate nucleoside is linked to the penultimate nucleoside via said reverse bond, and wherein (1) the reverse bond is a 5-5' reverse bond, and when reading toward the end comprising the terminal and penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 3'-5'; or (2) The reverse bond is a 3-3' reverse bond, and when reading toward the end including the terminal and the penultimate abasic nucleoside, the bond between the terminal and the penultimate abasic nucleoside is 5'-3'.

14. The inhibitor or the inhibitor for use according to claim 12 or 13, wherein the reverse internucleoside bond is located in the terminal region distal to the 5' terminal region of the second chain, or in the terminal region distal to the 3' terminal region of the second chain.

15. The inhibitor or the inhibitor for use according to any one of claims 12 to 14, wherein the inverted internucleoside bond is a 3'-3 inverted bond.

16. The inhibitor or the inhibitor for use according to any one of claims 12 to 14, wherein the reversed internucleoside bond is a 5'-5 reversed bond.

17. The inhibitor or the inhibitor for use according to any one of claims 1 to 16, wherein one or more nucleosides on the first strand and / or the second strand are modified to form modified nucleosides.

18. The inhibitor according to claim 17, or the inhibitor for use therefor, wherein the modification is a modification on the 2'-OH group of the ribose sugar, optionally selected from 2'-Me or 2'-F modifications.

19. The inhibitor according to claim 17 or 18, or the inhibitor for use thereof, wherein counting from position 1 of the first strand, the first strand comprises 2'-F at position 14, position 2, position 6 or any combination thereof.

20. The inhibitor or the inhibitor for use according to any one of claims 17-18, 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.

21. The inhibitor or the inhibitor for use according to any one of claims 17 to 20, wherein the first strand and the second strand each comprise a 2'-Me and a 2'-F modification.

22. The inhibitor according to any one of claims 17 to 21 or the inhibitor for use thereof, which is siRNA, wherein Suitably, the siRNA comprises at least one thermally labile modification at one or more positions in positions 1-9 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 1-9 of the first strand, wherein the labile modification is selected from modified unlocked nucleic acids (UNA) and glycol nucleic acids (GNA), preferably glycol nucleic acids.

23. The inhibitor according to claim 22 or the inhibitor for use therein, wherein The siRNA comprises at least one thermolabile modification at position 7 of the first strand, counting from position 1 of the first strand.

24. The inhibitor according to any one of claims 17 to 23 or the inhibitor for use therein, which is siRNA, wherein Counting from position 1 of the second strand, the siRNA comprises 3 or more 2'-F modifications at positions 7-13 of the second strand, such as positions 7-13 of the second strand comprise 4, 5, 6 or 7 2'-F modifications.

25. The inhibitor according to any one of claims 17 to 24 or the inhibitor for use thereof, which is siRNA, wherein Counting from position 1 of the second strand, the second strand comprises at least 3, such as 4, 5 or 6 2'-Me modifications at positions 1-6 of the second strand.

26. The inhibitor or the inhibitor for use according to any one of claims 17 to 25, which is siRNA, wherein the first strand comprises at least 5 consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside of the 3' terminal region, or at least within 1 or 2 nucleosides from the terminal nucleoside in the 3' terminal region.

27. The inhibitor or the inhibitor for use according to any one of claims 17 to 26, which is siRNA, wherein the first strand comprises 7 consecutive 2'-Me modifications in the 3' terminal region, preferably including the terminal nucleoside of the 3' terminal region.

28. The inhibitor according to any one of claims 17 to 23, or the inhibitor for use thereof, which is a siRNA, wherein the modified nucleosides of the second strand comprise a modification pattern (5'-3') according to any one of the following: (Me)8–(F)3–(Me) 10 。 29. The inhibitor or the inhibitor for use according to any one of claims 17 to 23 or 28, which is a siRNA, wherein the nucleosides of the first strand comprise 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 4 or 6 2'F modifications.

30. The inhibitor or the inhibitor for use according to any one of claims 17-23, or 28-29, which is a siRNA, wherein the modifications are at least selected from 2'Me and 2'F sugar modifications, wherein the total number of 2'F sugar modifications in the first strand consists of 3, 5 or 7 2'F modifications.

31. The inhibitor or the inhibitor for use according to any one of claims 17 to 30, wherein the siRNA oligomer further comprises one or more phosphorothioate internucleoside bonds.

32. An inhibitor according to claim 31 or an inhibitor for use thereof, wherein the one or more phosphorothioate internucleoside bonds are located between at least three consecutive positions of the 5' or 3' proximal terminal region of the second strand, respectively, whereby the proximal terminal region is preferably adjacent to the terminal region, wherein the one or more abasic nucleosides of the second strand according to at least claim 12 are located in the terminal region.

33. An inhibitor according to claim 31 or 32 or an inhibitor for use thereof, wherein the one or more phosphorothioate internucleoside bonds are respectively located between at least three consecutive positions in the 5' and / or 3' terminal region of the first strand, whereby preferably the terminal positions in the 5' and / or 3' terminal region of the first strand are connected to their adjacent positions via phosphorothioate internucleoside bonds.

34. The inhibitor according to any one of claims 11 to 33, or an inhibitor for use thereof, wherein the oligomer is a siRNA, the second strand of the siRNA being coupled directly or indirectly to one or more ligand moieties, wherein the ligand moiety is usually present in the terminal region of the second strand, preferably in its 3' terminal region.

35. The inhibitor according to claim 34 or the inhibitor for use thereof, wherein the ligand portion comprises i) one or more GalNAc ligands; and / or ii) one or more GalNAc ligand derivatives; and / or iii) one or more GalNAc ligands and / or GalNAc ligand derivatives coupled to the siRNA via a linker.

36. The inhibitor according to claim 35 or the inhibitor for use thereof, wherein the one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly coupled to the 5' or 3' terminal region of the second strand of the siRNA oligomer, preferably to the 3' terminal region thereof.

37. The inhibitor according to claim 35 or 36, or the inhibitor for use thereof, wherein the ligand portion comprises 38. The inhibitor according to claim 35 or 36, or the inhibitor for use thereof, having the following structure: in: R1, at each occurrence, is independently selected from the group consisting of hydrogen, methyl and ethyl; R2 is selected from the following group: hydrogen, hydroxyl, -OC 1-3 Alkyl, -C(=O)OC 1-3 Alkyl, halogen, and nitro; X1 and X2 at each occurrence are independently selected from the group consisting of methylene, oxygen and sulfur; m is an integer from 1 to 6; n is an integer from 1 to 10; q, r, s, t, v are independently integers from 0 to 4, provided that: (i) q and r cannot be zero at the same time; and (ii) s, t and v cannot all be 0 at the same time; Z is an oligomer.

39. The inhibitor according to claim 35 or 36, or the inhibitor for use thereof, having the following structure: in: r and s are independently integers selected from 1-16; and Z is an oligomer.

40. The inhibitor according to one or more of the preceding claims, or the inhibitor for use as described above, formulated as a pharmaceutical composition with excipients and / or carriers.

41. A pharmaceutical composition comprising an inhibitor according to one or more of the preceding claims, and a pharmaceutically acceptable excipient or carrier.

42. A pharmaceutical composition comprising an inhibitor according to one or more of the preceding claims, and a pharmaceutically acceptable excipient or carrier, for use in the treatment of diseases associated with coagulation disorders, such as hemophilia.

43. Use of ZPI as a target in identifying one or more therapeutic agents for treating a disease associated with coagulation disorders, such as hemophilia.

44. A method of treating or preventing a disease associated with a coagulation disorder, such as hemophilia, comprising administering to a patient a ZPI inhibitor, such as an inhibitor as defined in one or more of the preceding claims.

45. Use of ZPI as a biomarker for diseases associated with coagulation disorders, such as hemophilia.

46. ​​Use of a ZPI in an in vivo method for predicting susceptibility to a disease associated with a coagulation disorder, such as hemophilia, typically by monitoring the sequence and / or expression level and / or function of the ZPI in a sample obtained from a patient.

47. A method of predicting susceptibility to a disease associated with a coagulation disorder, such as hemophilia, and optionally treating a patient for a disease associated with a coagulation disorder, such as hemophilia, the method comprising: (a) obtaining a sample from a patient, (b) detecting the sequence and / or expression and / or function of the ZPI in said sample obtained from the patient, (c) predicting susceptibility to a disease associated with a coagulation disorder, such as hemophilia, based on the sequence and / or expression and / or function of the ZPI in said sample obtained from the patient, (d) preferably administering an effective amount of a ZPI inhibitor to the diagnosed patient.

48. Use of an inhibitor or composition according to any preceding claim in the preparation of a medicament for the treatment of a disease associated with coagulation disorders such as haemophilia.

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