Inhibitor of expression and / or function

Abstract

The present invention relates to inhibitors, compositions containing the inhibitors, and their use in the treatment or prevention of diseases related to hemostatic disorders such as hemophilia.

Description

Technical Field

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

Background Art

[0002] Inhibitors such as oligonucleoside / oligonucleotide compounds, which are inhibitors of gene expression and / or the expression or function of other targets such as lncRNA, can have important therapeutic applications in medicine. Oligonucleotides / oligonucleosides can be used to silence genes that are the cause of specific diseases. Gene silencing prevents protein formation 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 oligonucleoside / oligonucleotide that prevent protein formation by gene silencing.

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

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

Summary of the Invention

Means for Solving the Problems

[0005] The present invention is defined in the claims and relates in particular to the following. In one aspect, the present invention relates to an inhibitor of the expression and / or function of ZPI, which is conjugated to one or more ligand moieties.

[0006] In a further aspect, the present invention relates to an inhibitor according to the present invention, which is an siRNA oligomer.

[0007] In another aspect, the present invention relates to an inhibitor of the expression and / or function of ZPI, wherein the inhibitor is an siRNA oligomer.

[0008] In a further aspect, the present invention relates to an inhibitor according to the present invention, which comprises an siRNA oligomer conjugated to one or more ligand moieties.

[0009] In a further aspect, the present invention relates to an inhibitor according to the present invention for use in the prevention or treatment of a disease related to a hemostatic disorder such as hemophilia.

[0010] In a further aspect, the present invention relates to an inhibitor according to the present invention, wherein the one or more ligand moieties comprise one or more GalNAc ligands or one or more GalNAc ligand derivatives.

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

[0012] In a further aspect, the present invention relates to an inhibitor for use according to the present invention, wherein the target of the inhibitor is ZPI.

[0013] In a further aspect, the present invention relates to an siRNA oligomer having a first strand and a second strand, i) the first strand of the siRNA has a length in the range of 15 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 23 or 25, even more preferably 23, and / or ii) The second strand of the siRNA has a length in the range of 15 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 21 nucleosides, and relates to an inhibitor according to the invention or an inhibitor for use, which is an siRNA oligomer.

[0014] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the second sense strand further comprises one or more abasic nucleosides in the terminal region of the second strand, and the abasic nucleosides are connected to adjacent nucleosides via inverse nucleoside linkages.

[0015] In a further aspect, the second strand of the invention i) two or more abasic nucleosides in the terminal region of the second strand, and / or ii) two or more abasic nucleosides in either the 5' or 3' terminal region of the second strand, and / or iii) two or more abasic nucleosides in either the 5' or 3' terminal region of the second strand, which are present in a protruding manner as described herein, and / or iv) two or more consecutive abasic nucleosides in the terminal region of the second strand, preferably one such abasic nucleoside is the terminal nucleoside, two or more consecutive abasic nucleosides, and / or v) two or more consecutive abasic nucleosides in either the 5' or 3' terminal region of the second strand, preferably one such abasic nucleoside is the terminal nucleoside of either the 5' or 3' terminal region of the second strand, two or more consecutive abasic nucleosides, and / or vi) an inverse nucleoside linkage connecting at least one abasic nucleoside to an adjacent base nucleoside in the terminal region of the second strand, and / or vii) an inverse internucleoside linkage connecting at least one abasic nucleoside to an adjacent nucleoside at either the 5' or 3' terminal region of the second strand, and / or viii) an abasic nucleoside as the penultimate nucleoside connected via an inverse linkage to a nucleoside that is not the terminal nucleoside (referred to herein as the third last nucleoside), and / or ix) an abasic nucleoside as two terminal nucleosides connected via a 5'-3' linkage when reading the strand in the direction towards its terminus, x) an abasic nucleoside as two terminal nucleosides connected via a 3'-5' linkage when reading the strand in the direction towards the terminus containing the terminal nucleoside, xi) abasic nucleosides at the two terminal positions, wherein the penultimate nucleoside is connected to the third last nucleoside via an inverse linkage, which is a 5-5' inverse linkage or a 3'-3' inverse linkage, xii) abasic nucleosides at the two terminal positions, wherein the penultimate nucleoside is connected to the third last nucleoside via an inverse linkage, (1) the inverse linkage is a 5-5' inverse linkage, and the linkage between the terminus and the penultimate abasic nucleoside is 3'5' when reading towards the terminus containing the terminus and the penultimate abasic nucleoside, or (2) the inverse linkage is a 3-3' inverse linkage, and the linkage between the terminus and the penultimate abasic nucleoside is 5'3' when reading towards the terminus containing the terminus and the penultimate abasic nucleoside, any of which is an abasic nucleoside relates to an inhibitor according to the invention or an inhibitor for use according to the invention.

[0016] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein the inverse internucleoside linkage is present in a terminal region distal from the 5' terminal region of the second strand or in a terminal region distal from the 3' terminal region of the second strand.

[0017] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the reverse nucleoside internucleoside linkage is a 3'3 reverse linkage.

[0018] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the reverse nucleoside internucleoside linkage is a 5'5 reverse linkage.

[0019] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein one or more nucleosides of the first strand and / or the second strand are modified to form modified nucleosides.

[0020] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the modification is a modification at the 2'-OH group of the ribose sugar and is optionally selected from a 2'-Me modification or a 2'-F modification.

[0021] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the first strand contains 2'-F at the 14th, 2nd, 6th position, or any combination thereof, counted from the 1st position of the first strand.

[0022] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the second strand contains a 2'-F modification at the 7th, and / or 9th, and / or 11th, and / or 13th position, counted from the 1st position of the second strand.

[0023] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the first strand and the second strand each contain a 2'-Me modification and a 2'-F modification.

[0024] In a further aspect, the present invention relates to an siRNA, which preferably contains at least one heat destabilizing modification at one or more positions from position 1 to position 9 of the first strand, counting from position 1 of the first strand, and / or at one or more positions of the second strand aligned with positions 1 to 9 of the first strand. The destabilizing modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), and is preferably glycol nucleic acid, and relates to an inhibitor according to the present invention or an inhibitor for use.

[0025] In a further aspect, the present invention relates to an inhibitor according to the present invention or an inhibitor for use, wherein the siRNA contains at least one heat destabilizing modification at position 7 of the first strand, counting from position 1 of the first strand.

[0026] In a further aspect, the present invention relates to an siRNA, and the siRNA contains 3 or more 2'-F modifications at positions 7 to 13 of the second strand, counting from position 1 of the second strand, for example, 4, 5, 6, or 7 2'-F modifications at positions 7 to 13 of the second strand, and relates to an inhibitor according to the present invention or an inhibitor for use.

[0027] In a further aspect, the present invention relates to an siRNA, and the second strand contains at least 3, for example, 4, 5, or 6 2'-Me modifications at positions 1 to 6 of the second strand, counting from position 1 of the second strand, and relates to an inhibitor according to the present invention or an inhibitor for use.

[0028] In a further aspect, the present invention relates to an siRNA, and the first strand preferably contains at least 5 consecutive 2'-Me modifications in the 3'-terminal region, including the terminal nucleoside of the 3'-terminal region, or within at least one or two nucleosides from the terminal nucleoside of the 3'-terminal region, and relates to an inhibitor according to the present invention or an inhibitor for use.

[0029] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, which is an siRNA, wherein the first strand preferably contains 7 consecutive 2'-Me modifications in the 3' terminal region, including the terminal nucleoside of the 3' terminal region.

[0030] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the siRNA oligomer further comprises one or more phosphorothioate internucleoside linkages.

[0031] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein each of the one or more phosphorothioate internucleoside linkages is present between at least 3 consecutive positions in the 5' or 3' near-terminal region of the second strand, and the near-terminal region is preferably adjacent to the terminal region where one or more abasic nucleosides of the second strand are located as defined herein.

[0032] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein each of the one or more phosphorothioate internucleoside linkages is present between at least 3 consecutive positions in the 5' and / or 3' terminal region of the first strand, and preferably the terminal positions of the 5' and / or 3' terminal region of the first strand are attached to their adjacent positions by a phosphorothioate internucleoside linkage.

[0033] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the oligomer is an siRNA, and the second strand of the siRNA is directly or indirectly conjugated to one or more ligand moieties, and the ligand moiety is typically present in the terminal region of the second strand, preferably in its 3' terminal region.

[0034] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, wherein the ligand moiety is i) one or more GalNAc ligands, and / or ii) one or more GalNAc ligand derivatives, and / or iii) one or more GalNAc ligands and / or GalNAc ligand derivatives conjugated to said siRNA via a linker relates to an inhibitor according to the invention or an inhibitor for use according to the invention, comprising

[0035] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein said one or more GalNAc ligands and / or GalNAc ligand derivatives are conjugated directly or indirectly to the 5' or 3' end region of the second strand of the siRNA oligomer, preferably to its 3' end region.

[0036] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use according to the invention, wherein the ligand moiety comprises

[0037]

Chemical formula

[0038] In a further aspect, the invention relates to a structure:

[0039]

Chemical formula

[0040] In a further aspect, the invention relates to a structure:

[0041]

Chemical formula

[0042] In a further aspect, the invention relates to an inhibitor according to the invention or an inhibitor for use, formulated as a pharmaceutical composition having an excipient and / or a carrier.

[0043] In another aspect, the invention relates to a pharmaceutical composition comprising an inhibitor according to one or more of the preceding aspects in combination with a pharmaceutically acceptable excipient or carrier.

[0044] In a further aspect, the invention relates to a pharmaceutical composition comprising an inhibitor according to the invention in combination with a pharmaceutically acceptable excipient or carrier for use in the treatment of a disease associated with a hemostatic disorder such as hemophilia.

[0045] In another aspect, the invention relates to the use of ZPI as a target for identifying one or more therapeutic agents for treating a disease associated with a hemostatic disorder such as hemophilia.

[0046] In another aspect, the present invention relates to a method for treating or preventing a disease related to a hemostatic disorder such as hemophilia, the method comprising administering to a patient an inhibitor of ZPI, such as an inhibitor defined by one or more of the preceding aspects.

[0047] In another aspect, the present invention relates to ZPI for use as a biomarker for a disease related to a hemostatic disorder such as hemophilia.

[0048] In another aspect, the present invention relates to ZPI for use in an in vivo method for predicting susceptibility to a disease related to a hemostatic disorder such as hemophilia, typically by monitoring the level of the sequence and / or expression and / or function of ZPI in a sample obtained from a patient.

[0049] In another aspect, the present invention is a method for predicting a patient's susceptibility to a disease related to a hemostatic disorder such as hemophilia and optionally treating a disease related to a hemostatic disorder such as hemophilia, the method comprising: (a) obtaining a sample from the patient, (b) detecting the sequence and / or expression and / or function of ZPI in the sample obtained from the patient, (c) predicting susceptibility to a disease related to a hemostatic disorder such as hemophilia based on the sequence and / or expression and / or function of ZPI in the sample obtained from the patient, (d) preferably, administering an effective amount of a ZPI inhibitor to the diagnosed patient and related to a method comprising.

[0050] In another aspect, the present invention relates to an inhibitor or composition according to the present invention in the preparation of a medicament for use in the treatment of a disease related to a hemostatic disorder such as hemophilia. BRIEF DESCRIPTION OF THE DRAWINGS

[0051]

Figure 1a

Figure 1b

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6-1

Figure 6-2

Figure 6-3

Figure 6-4

Figure 6-5

Figure 6-6

Figure 6-7

Figure 6-8

Figure 7-1

Figure 7-2

Figure 7-3

Figure 7-4

Figure 7-5

Figure 8A

Figure 8B

Figure 9-1

Figure 9-2

Figure 10A

Figure 10B

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 12C

Figure 12D

Figure 12E

Figure 12F

Figure 12G

Figure 12H

Figure 12I

Figure 12J

Figure 12K

Figure 12L

Figure 12M

Mode for Carrying Out the Invention

[0052] The present invention provides, inter alia, inhibitors capable of affecting the expression of a target by binding to mRNA transcribed from a gene, oligomers such as nucleic acids, such as inhibitory RNA molecules (which may be referred to as iRNA or siRNA), and compositions containing them. The target may be present intracellularly, for example, within cells in a subject such as a human. The inhibitor can be used, for example, for the prevention and / or treatment of medical conditions associated with the expression of the target gene.

[0053] In particular, the present invention identifies inhibitors of ZPI that are useful for the prevention and / or treatment of diseases related to hemostatic disorders such as hemophilia.

[0054] Protein Z-dependent protease inhibitor (ZPI) is a protein that circulates in the blood and inhibits factor Xa and factor XIa of the coagulation cascade. This protein belongs to the class of serine protease inhibitors (serpins). ZPI is encoded in humans by the ZPI gene (SEQ ID NO: 1).

[0055] SEQ ID NO: 1 (ZPI)

[0056] The inventors used network analysis to assign multiple genes or proteins to fewer driver processes and to identify potent drug targets from these processes. This approach utilizes information that is typically ignored in standard gene set analysis, namely, known and predicted interactions between genes (and proteins) and the inclusion of other genes in the same or related pathways. In particular, the inventors analyzed the human blood coagulation process using a network model in which ZPI is highlighted as a preferred target for hemophilia, and antithrombin 3 (AT3, the target of Futhan), an existing drug target, is also highlighted, thereby validating this network approach. Experimental confirmation that inhibition of ZPI is indeed a promising strategy for treating hemophilia is provided in Example 9.

[0057] The inhibition disclosed herein may be an inhibition of a gene or a protein resulting from the expression of the gene, and reference to ZPI thereby expressly incorporates reference to inhibition of the expression or function of the gene and separately the protein product.

[0058] Definitions The "first strand" is also referred to herein as the antisense strand or the guide strand, which can be used interchangeably herein, and refers to a nucleic acid strand, such as an siRNA, such as a strand of dsiRNA, that contains a region that is substantially complementary to a target sequence, such as an mRNA. As used herein, the term "region of complementarity" refers to a region of the antisense strand that is substantially complementary to a sequence, such as a target sequence. If the region of complementarity is not completely complementary to the target sequence, the mismatch may be in the internal region or the terminal region of the molecule. In some embodiments, the double-stranded nucleic acid of the invention, such as an siRNA agent, contains nucleotide mismatches in the antisense strand.

[0059] The "second strand" (also referred to herein as the sense strand or the passenger strand, which can be used interchangeably herein) refers to a nucleic acid strand, such as a strand of siRNA, that contains a region that is substantially complementary to the region of the antisense strand as the term is defined herein.

[0060] In the context of a molecule comprising a nucleic acid having a ligand moiety and optionally also a linker moiety, the nucleic acids of the invention may be referred to as an oligonucleotide moiety or an oligonucleoside moiety.

[0061] Oligonucleotides are short nucleic acid polymers. Oligonucleotides contain phosphodiester bonds between their nucleoside components (base + sugar), but the invention is not limited to oligonucleotides that are always joined by such phosphodiester bonds between adjacent nucleosides, and other oligomers of nucleosides joined by bonds other than phosphate bonds are contemplated. For example, the bond between nucleotides may be a phosphorothioate bond. Thus, the term "oligonucleoside" herein encompasses both oligonucleotides and other oligomers of nucleosides. According to the invention, oligonucleosides which are nucleic acids having at least a portion that is an oligonucleotide are preferred. According to the invention, oligonucleosides having one or more or most of the phosphodiester backbone bonds between nucleosides are also preferred. According to the invention, oligonucleosides having one or more or most of the phosphodiester backbone bonds between nucleosides and also having one or more phosphorothioate backbone bonds between nucleosides (typically in the terminal regions of the first strand and / or the second strand) are also preferred.

[0062] In some embodiments, the double-stranded nucleic acids of the invention, such as siRNA agents, contain nucleoside mismatches in the sense strand. In some embodiments, the nucleoside mismatches are present, for example, within 5, 4, 3, 2, or 1 nucleoside from the 3' end of the nucleic acid, such as siRNA.

[0063] In another embodiment, the nucleoside mismatch is present, for example, at the 3'-terminal nucleoside of a nucleic acid, such as siRNA.

[0064] The "target sequence" (which may be referred to as target RNA or target mRNA) refers to a contiguous portion of the nucleoside sequence of an mRNA molecule formed during gene transcription, including the mRNA that is the product of RNA processing of the primary transcript, or may be a contiguous portion of the nucleotide sequence of any RNA molecule, such as an lncRNA, whose inhibition is desired.

[0065] The target sequence may be about 10 to 35 nucleosides in length, for example about 15 to 30 nucleosides in length. For example, the target sequence may be 15 to 30 nucleosides in length, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleosides in length. Ranges and lengths intermediate to those listed above are also contemplated as being part of the present invention.

[0066] The term "ribonucleoside" or "nucleoside" can also refer to a modified nucleoside, as will be described in more detail below.

[0067] The nucleic acid may be DNA or RNA and may contain modified nucleosides. A preferred nucleic acid is RNA.

[0068] As used interchangeably herein, the terms "iRNA", "siRNA", "RNAi agent", and "iRNA agent", "RNA interference agent" refer to agents that contain RNA and mediate the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. siRNA directs the sequence-specific degradation of mRNA by RNA interference (RNAi).

[0069] Double-stranded RNA is referred to herein as "double-stranded siRNA (dsiRNA) agent", "double-stranded siRNA (dsiRNA) molecule", "double-stranded RNA (dsRNA) agent", "double-stranded RNA (dsRNA) molecule", "dsiRNA agent", "dsiRNA molecule", or "dsiRNA", and refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands that are said to have a "sense" direction and an "antisense" direction with respect to the target RNA. Most of the nucleosides of each strand of the nucleic acid, for example, the dsRNA molecule, are preferably ribonucleosides, but in that case, each strand or both strands may further contain one or more non-ribonucleosides, such as deoxyribonucleosides or modified ribonucleosides. In addition, as used herein, "siRNA" may contain ribonucleosides having chemical modifications.

[0070] The term "modified nucleoside" independently refers to a nucleoside having a modified sugar moiety, a modified internucleoside linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleoside includes substitutions, additions, or removals to the internucleoside linkage, sugar moiety, or nucleobase, such as functional groups or atoms. Any such modifications used in siRNA-type molecules are included within "iRNA" or "RNAi agent" or "siRNA" or "siRNA agent" for the purposes of this specification and the claims.

[0071] The double-stranded region of the nucleic acid of the present invention, such as dsRNA, is about 9 to 40 base pairs in length, for example 9 to 36 base pairs in length, for example, about 15 to 30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length.

[0072] The two strands forming the double-stranded structure may be different parts of one larger molecule or may be separate molecules, such as RNA molecules.

[0073] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the double-stranded structure of a double-stranded nucleic acid. The ds nucleic acid may contain an overhang of at least one nucleoside, or alternatively, the overhang may contain at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more nucleosides. The nucleoside overhang may contain or consist of a nucleoside analog containing a deoxynucleoside. The overhang may be present in the sense strand, the antisense strand, or any combination thereof. Further, the overhanging nucleosides may be present at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand.

[0074] In certain embodiments, the antisense strand has a 1-10 nucleoside overhang, for example, a 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside overhang at the 3' or 5' end.

[0075] "Blunt" or "blunt end" means that there are no unpaired nucleosides at the ends of the double-stranded nucleic acid, i.e., no nucleoside overhangs. Nucleic acids of the present invention include those having no nucleoside overhang at one end or no nucleoside overhangs at either end.

[0076] Unless otherwise indicated, the term "complementary" is used to describe a first nucleoside sequence in relation to a second nucleoside sequence, and when used in this context, an oligonucleoside containing the first nucleoside sequence is capable of hybridizing under certain conditions to an oligonucleoside or polynucleotide containing the second nucleoside sequence to form a double-stranded structure, as would be understood by one of ordinary skill in the art. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al. (1989) Cold Spring Harbor Laboratory Press).

[0077] The complementary sequences in the nucleic acids described herein, e.g., in dsiRNA, involve base pairing over the entire length of one or both nucleoside sequences of an oligonucleoside or polynucleotide containing a first nucleoside sequence and an oligonucleoside or polynucleotide containing a second nucleoside sequence. Such sequences can be referred to herein as "perfectly complementary" to each other. However, herein, when a first sequence is referred to as "substantially complementary" or "partially complementary" to a second sequence, the two sequences may be perfectly complementary or form one or more, but preferably no more than 5 mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, while retaining the ability to hybridize under conditions most relevant to the ultimate application, e.g., inhibition of gene expression via the RISC pathway. Bulges shall not be considered mismatches with respect to the determination of complementarity. Further, for example, a nucleic acid, e.g., dsRNA, containing one oligonucleoside 17 nucleosides in length and another oligonucleoside 19 nucleosides in length, wherein the longer oligonucleoside contains a 17-nucleoside sequence that is perfectly complementary to the shorter oligonucleoside, can be called "perfectly complementary".

[0078] Also, "complementary" sequences, as used herein, may or may only consist of base pairs formed from non-Watson-Crick base pairs or non-natural and modified nucleosides, as long as they meet the above requirements regarding their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogsteen base pairing.

[0079] The terms "complementary", "perfectly complementary", and "substantially / partially complementary" can be used herein with respect to base matching between the sense and antisense strands of a nucleic acid, e.g., dsiRNA, or between the antisense strand of a double-stranded nucleic acid, e.g., an siRNA agent, and a target sequence.

[0080] Within the present invention, the second strand of the nucleic acid according to the present invention, particularly the dsiRNA for inhibiting ZPI, is at least partially complementary to the first strand of the nucleic acid. In certain embodiments, the first and second strands of the nucleic acid according to the present invention have a length of at least 17 base pairs and are partially complementary when forming a double-stranded region containing 1, 2, 3, 4, or 5 or fewer mismatched base pairs.

[0081] In certain embodiments, the first and second strands of the nucleic acid according to the present invention have a length of 19 base pairs and are partially complementary when forming a double-stranded region having 1, 2, 3, 4, or 5 or fewer mismatched base pairs. In certain embodiments, the first and second strands of the nucleic acid according to the present invention have a length of 21 base pairs and are partially complementary when forming a double-stranded region having 1, 2, 3, 4, or 5 or fewer mismatched base pairs.

[0082] Alternatively, the first and second strands of the nucleic acid according to the present invention are a double-stranded region having a length of at least 17 base pairs, wherein at least 14, 15, 16, or 17 of the base pairs are complementary base pairs, particularly Watson-Crick base pairs, and are partially complementary when forming a double-stranded region.

[0083] In certain embodiments, the first and second strands of the nucleic acid according to the present invention form a double-stranded region having a length of 19 base pairs, wherein at least 14, 15, 16, 17, 18, or all 19 base pairs are complementary base pairs, particularly Watson-Crick base pairs, and are partially complementary when forming the double-stranded region. In certain embodiments, the first and second strands of the nucleic acid according to the present invention form a double-stranded region having a length of 21 base pairs, wherein at least 16, 17, 18, 19, 20, or all 21 base pairs are complementary base pairs, particularly Watson-Crick base pairs, and are partially complementary when forming the double-stranded region. As used herein, a nucleic acid that is "substantially complementary" or "partially complementary" to at least a portion of messenger RNA (mRNA) refers to a polynucleotide that is substantially or partially complementary to a continuous portion of the mRNA of interest (e.g., mRNA encoding a gene). In certain embodiments, the continuous portion of the mRNA is one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2 to 121. For example, a polynucleotide is complementary to at least a portion of the mRNA of the gene of interest if its sequence is substantially or partially complementary to an uninterrupted portion of the mRNA encoding that gene.

[0084] Thus, in some preferred embodiments, the antisense oligonucleotides disclosed herein are completely complementary to the target gene sequence.

[0085] In other embodiments, the antisense oligonucleotides disclosed herein are substantially or partially complementary to the target RNA sequence and are at least about 80% complementary over their entire length to an equivalent region of the target RNA sequence, e.g., at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, and include a continuous nucleoside sequence.

[0086] In certain embodiments, the first (antisense) strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of the RNA transcribed from the ZPI gene. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of at least 17 nucleosides of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleosides of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention is partially or fully complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleosides of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2 to 121.

[0087] In certain embodiments, the first (antisense) strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of at least 17 nucleosides, and at least 14, 15, 16, or 17 nucleosides of said continuous nucleoside sequence are partially complementary to a continuous portion of the ZPI mRNA when they are complementary to a continuous portion of the ZPI mRNA. In certain embodiments, the first strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of at least 17 nucleosides, and at least 14, 15, 16, or 17 nucleosides of said continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2 to 121. In certain embodiments, the first strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of 19 nucleosides, and at least 14, 15, 16, 17, 18, or all 19 of said continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2 to 121. In certain embodiments, the first strand of the nucleic acid according to the invention comprises a continuous nucleoside sequence of 23 nucleosides, and at least 18, 19, 20, 21, 22, or all 23 of said continuous nucleoside sequence are complementary to a continuous portion of any one of the sequences listed in Table 1, i.e., any one of SEQ ID NOs: 2 to 101.

[0088] In some embodiments, a nucleic acid of the invention, such as siRNA, comprises a sense strand that is substantially or partially complementary to an antisense polynucleoside, and the antisense polynucleoside is, in turn, complementary to a target gene sequence, and is at least about 80% complementary over the entire length to an equivalent region of the nucleoside sequence of the antisense strand, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, and comprises a continuous nucleoside sequence.

[0089] In some embodiments, a nucleic acid of the invention, such as siRNA, comprises an antisense strand that is substantially or partially complementary to a target sequence, and is at least 80% complementary over the entire length to the target sequence, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, and comprises a continuous nucleoside sequence.

[0090] As used herein, a "subject" is an animal, including a mammal (such as a primate (human, non-human primate such as a monkey and chimpanzee, etc.) or non-primate) or a bird, that expresses an endogenous or heterologous target gene when the target gene sequence has sufficient complementarity to promote target knockdown against a nucleic acid, such as an iRNA agent. In certain preferred embodiments, the subject is human.

[0091] The terms "treat" or "treatment" refer to beneficial or desired results, including, but not limited to, alleviation or amelioration of one or more symptoms associated with gene expression. "Treatment" may also mean extending the survival period as compared to the expected survival period without treatment. Treatment may include prevention of the onset of complications, e.g., reduction of liver damage in a subject having a liver infection.

[0092] "Therapeutically effective amount", as used herein, when administered to a patient to treat a subject having a disease, is intended to include an amount of a nucleic acid, such as an iRNA, sufficient to achieve treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms or associated complications thereof).

[0093] The term "pharmaceutically acceptable" as used herein refers to compounds, substances, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without undue toxicity, irritation, allergic response, or other problems or complications and that are commensurate with a reasonable benefit / risk ratio.

[0094] The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject to be treated.

[0095] When values or ranges of values of parameters are recited, intermediate values and ranges within the recited values are also intended to be part of the present invention.

[0096] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article.

[0097] The term "comprising" is used herein to mean "including but not limited to" and is used interchangeably therewith.

[0098] As used herein, the term "or" means the term "and / or" and is used interchangeably therewith, unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand, or sense strand and antisense strand".

[0099] As used herein, the term "about" is used to mean within the typical ranges of error tolerance in the art. For example, "about" can be understood to be about two standard deviations from the average. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When "about" is present before a series of numbers or a range, it is understood that "about" can modify each of the numbers in that series of numbers or range.

[0100] The term "at least" before a number or 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 as apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleosides of a 21-nucleoside nucleic acid molecule" means that 18, 19, 20, or 21 nucleosides have the stated characteristics. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in that series of numbers or range.

[0101] As used herein, "below" or "less than" is understood to mean the value adjacent to this phrase, and, in theory, a lower value, or, in some cases, zero or an integer as logically determined by the context. For example, a duplex having an overhang "of 2 nucleosides or less" has an overhang of 2, 1, or 0 nucleosides. When "below" is present after a series of numbers or a range, it is understood that "below" can modify each of the numbers in that series of numbers or range.

[0102] The terminal region of a strand is the last 5 nucleotides from the 5' or 3' end.

[0103] The nucleobase sequence is the sequence of bases of an oligomeric nucleic acid.

[0104] The various embodiments of the present invention can be combined as determined to be appropriate by those skilled in the art.

[0105] Target The targets of inhibition disclosed herein may be, but are not limited to, mRNA, polypeptide, protein, or gene.

[0106] These targets are targets whose inhibition aids in the prevention or treatment of diseases related to hemostatic disorders such as hemophilia.

[0107] The target of inhibition is ZPI, and the inhibition can be achieved by inhibiting the expression or function of the ZPI gene or protein or both.

[0108] In a preferred embodiment, the target is mRNA expressed from the ZPI gene. Exemplary target sequences of ZPI mRNA are listed in Table 1 below.

[0109]

Table 1

[0110] It should be understood that SEQ ID NOs: 2 to 121 relate to human (Homo sapiens) mRNA sequences.

[0111] Disease / condition The present invention relates to inhibitors suitable for or for use in the treatment of diseases related to hemostatic disorders such as hemophilia.

[0112] Hemophilia is a mostly genetic genetic disorder that impairs the body's ability to perform blood clotting, a process necessary to stop bleeding. As a result, the bleeding time after an injury in the subject becomes longer, bruising is more likely, and the risk of bleeding within joints or the brain increases. Patients with mild cases of this disease may only show symptoms after an accident or during surgery. Bleeding within joints can cause permanent damage, and bleeding in the brain can cause long-term headaches, seizures, or a decrease in the level of consciousness.

[0113] There are two main types of hemophilia: hemophilia A, which occurs due to low levels of blood clotting factor VIII, and hemophilia B, which occurs due to low levels of blood clotting factor IX. These are typically inherited from both parents via the X chromosome with a non-functional gene. Rarely, new mutations can occur during early development, or hemophilia can develop later in life 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 pathway from functioning properly. This clotting pathway is necessary when the endothelium of blood vessels is damaged. Acquired hemophilia is associated with cancer, autoimmune diseases, and pregnancy. Diagnosis is by testing the blood's clotting ability and the levels of clotting factors.

[0114] In certain embodiments, the inhibitors of the present invention are suitable for or for the treatment of hemophilia A, B, and / or C. In certain embodiments, the inhibitors of the present invention are suitable for or for the treatment of hemophilia A and / or B. In certain embodiments, the inhibitors of the present invention are suitable for or for the treatment of acquired hemophilia. In certain embodiments, the inhibitors of the present invention are suitable for or for the treatment of von Willebrand disease. In certain embodiments, the inhibitors of the present invention are suitable for or for the treatment of parahemophilia.

[0115] While not wishing to be bound by theory, treatment with the inhibitors of the present invention results in an enhancement of coagulation factor levels and can reduce or prevent blood loss, as demonstrated in FIG. 10 herein. Thus, in a preferred embodiment, treatment with the inhibitors of the present invention reduces or prevents blood loss episodes in a subject suffering from hemophilia. In another preferred embodiment, treatment with the inhibitors of the present invention reduces or prevents intra-articular blood loss in a subject suffering from hemophilia. In certain embodiments, treatment with the inhibitors of the present invention reduces or prevents intramuscular or intracerebral blood loss in a subject suffering from hemophilia.

[0116] Alternatively or in addition, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with the inhibitors of the present invention can result in one or more of the following.

[0117] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with the inhibitors of the present invention results in a reduction of myeloproliferation. As shown in FIG. 12A, treatment of hemA mice with the inhibitors of the present invention significantly reduced myeloproliferation in said mice.

[0118] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with the inhibitors of the present invention results in a reduction of osteoarthritis. As shown in FIG. 12B, treatment of hemA mice with the inhibitors of the present invention significantly reduced osteoarthritis in said mice.

[0119] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with the inhibitors of the present invention results in a reduction of chondrocyte degeneration / necrosis. As shown in FIG. 12C, treatment of hemA mice with the inhibitors of the present invention significantly reduced chondrocyte degeneration / necrosis in said mice.

[0120] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction in bleeding. As shown in Figure 12D, treatment of hemA mice with an inhibitor of the present invention significantly reduced bleeding in said mice.

[0121] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction in hemosiderin deposition. As shown in Figure 12E, treatment of hemA mice with an inhibitor of the present invention significantly reduced hemosiderin deposition in said mice.

[0122] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction in hematoma formation. As shown in Figure 12F, treatment of hemA mice with an inhibitor of the present invention significantly reduced hematomas in said mice.

[0123] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction in osteoclastic bone resorption. As shown in Figure 12G, treatment of hemA mice with an inhibitor of the present invention significantly reduced osteoclastic bone resorption in said mice.

[0124] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction in osteolysis. As shown in Figure 12H, treatment of hemA mice with an inhibitor of the present invention significantly reduced osteolysis in said mice.

[0125] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction in periostitis. As shown in Figure 12I, treatment of hemA mice with an inhibitor of the present invention significantly reduced periostitis in said mice.

[0126] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction of subchondral sclerosis. As shown in FIG. 12J, treatment of hemA mice with an inhibitor of the present invention significantly reduced subchondral sclerosis in said mice.

[0127] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction of tendon degeneration. As shown in FIG. 12K, treatment of hemA mice with an inhibitor of the present invention significantly reduced tendon degeneration in said mice.

[0128] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction of tendinitis. As shown in FIG. 12L, treatment of hemA mice with an inhibitor of the present invention significantly reduced tendinitis in said mice.

[0129] In certain embodiments, treatment of a subject, preferably a subject having a hemostatic disorder such as hemophilia, with an inhibitor of the present invention results in a reduction of tenosynovitis. As shown in FIG. 12M, treatment of hemA mice with an inhibitor of the present invention significantly reduced tenosynovitis in said mice.

[0130] Accordingly, in certain embodiments, the present invention relates to an inhibitor suitable for use in the treatment of hemophilia or for use in treatment, wherein treatment of hemophilia is characterized by a reduction in blood loss and one or more of the following: a reduction in myeloproliferation, a reduction in osteoarthritis, a reduction in chondrocyte degeneration / necrosis, a reduction in bleeding, a reduction in hemosiderin deposition, a reduction in hematoma, a reduction in osteoclastic bone resorption, a reduction in osteolysis, a reduction in osteoperiostitis, a reduction in subchondral sclerosis, a reduction in tendon degeneration, a reduction in tendinitis, and / or a reduction in tenosynovitis.

[0131] As used herein, the term "treatment" refers to medical therapy for any human or other vertebrate subject in need thereof. The subject has undergone a physical examination by a physician or veterinarian, and a tentative or definitive diagnosis is expected to have been made indicating that the use of the particular treatment will be beneficial in 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 prophylactic, palliative, symptomatic, and / or curative.

[0132] Inhibitor Inhibitors of the present invention include nucleic acids such as siRNA, antibodies and antigen-binding fragments thereof, such as monoclonal antibodies, polypeptides, antibody-drug conjugates, and small molecules. Nucleic acids such as siRNA are preferred.

[0133] Certain preferred features of the inhibitors of the present invention, which are oligonucleosides such as siRNA, are shown below.

[0134] In certain embodiments, the nucleic acid comprises a first strand comprising a sequence that is at least partially complementary to a portion of the RNA transcribed from the ZPI gene (SEQ ID NO: 1). In preferred embodiments, the nucleic acid comprises a first strand comprising a sequence that is at least partially complementary to ZPI mRNA (NM_016186.3).

[0135] In certain embodiments, the nucleic acid for inhibiting the expression of ZPI comprises a double-stranded region comprising a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand (i) is at least partially complementary to a portion of the RNA transcribed from the ZPI gene, (ii) comprises at least 17 consecutive nucleosides that differ from any one of SEQ ID NOs: 122 to 241 by 0 or 1 nucleoside.

[0136] In certain embodiments, the first strand comprises nucleosides 2 to 18 of any one of the sequences shown in SEQ ID NOs: 122 to 241.

[0137] In certain embodiments, the first strand comprises any one of SEQ ID NOs: 122 to 241.

[0138] In certain embodiments, the nucleic acid for inhibiting the expression of ZPI comprises a double-stranded region including a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand (i) is at least partially complementary to a portion of the RNA transcribed from the ZPI gene, (ii) comprises at least 21 consecutive nucleosides that differ from any one of SEQ ID NOs: 122 to 241 by 0 or 1 nucleoside.

[0139] In certain embodiments, the first strand comprises nucleosides 2 to 22 of any one of the sequences shown in SEQ ID NOs: 122 to 241.

[0140] In certain embodiments, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of SEQ ID NOs: 242 to 361 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleosides.

[0141] In certain embodiments, the second strand comprises a nucleoside sequence of at least 19 consecutive nucleosides that differ from any one of SEQ ID NOs: 242 to 361 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 19 consecutive nucleosides.

[0142] In certain embodiments, the second strand comprises a nucleoside sequence of at least 21 consecutive nucleosides that differ from any one of SEQ ID NOs: 242 to 361 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 21 consecutive nucleosides.

[0143] In certain embodiments, the second strand comprises any one of SEQ ID NOs: 242 to 361.

[0144] In certain embodiments, the nucleic acid comprises, consists of, or consists essentially of a first strand having a nucleoside sequence that differs from any one of SEQ ID NOs: 122 to 241 by 0 or 1 nucleoside, and a second strand having a nucleoside sequence that differs from any one of SEQ ID NOs: 242 to 361 by 0 or 1 nucleoside.

[0145] As used herein, the double-stranded region is preferably formed between a first (antisense) strand and a complementary second (sense) strand. Exemplary pairs of complementary antisense and sense strands are listed in Table 2 below.

[0146] [Table 2] JPEG2025519224000008.jpg243148JPEG2025519224000009.jpg243149JPEG2025519224000010.jpg243148JPEG2025519224000011.jpg66146

[0147] In certain embodiments, the invention relates to a nucleic acid comprising, consisting of, or consisting essentially of a first strand and a second strand having a nucleoside sequence that differs from any one of the following first and second sequences by 0 or 1 nucleoside.

[0148] [Table 3]

[0149] In certain embodiments, the nucleic acid for inhibiting the expression of ZPI comprises a double-stranded region comprising a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand is (i) at least partially complementary to a portion of the RNA transcribed from the ZPI gene, (ii) It contains at least 17 consecutive nucleosides that differ by 0 or 1 nucleoside from any one of SEQ ID NOs: 362 to 561, 762 to 771, 782 to 786, or 795 to 797.

[0150] In certain embodiments, the first strand contains nucleosides 2 to 18 of any one of the sequences shown in SEQ ID NOs: 362 to 561, 762 to 771, 782 to 786, or 795 to 797.

[0151] In certain embodiments, the nucleic acid for inhibiting the expression of ZPI comprises a double-stranded region containing a first strand and a second strand that is at least partially complementary to the first strand, wherein the first strand (i) is at least partially complementary to a portion of the RNA transcribed from the ZPI gene, (ii) contains at least 21 consecutive nucleosides that differ by 0 or 1 nucleoside from any one of SEQ ID NOs: 362 to 561, 762 to 771, 782 to 786, or 795 to 797.

[0152] In certain embodiments, the first strand contains nucleosides 2 to 22 of any one of the sequences shown in SEQ ID NOs: 362 to 561, 762 to 771, 782 to 786, or 795 to 797.

[0153] In certain embodiments, the first strand contains any one of SEQ ID NOs: 362 to 561, 762 to 771, 782 to 786, or 795 to 797.

[0154] The modification patterns of the nucleic acids shown in SEQ ID NOs: 362 to 561, 762 to 771, 782 to 786, or 795 to 797 are summarized in Table 3 below.

[0155]

Table 4

[0156] In certain embodiments, the second strand comprises a nucleoside sequence of at least 17 consecutive nucleosides that differs from any one of SEQ ID NOs: 562-761, 772-781, or 798-800 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 17 consecutive nucleosides.

[0157] In certain embodiments, the second strand comprises a nucleoside sequence of at least 19 consecutive nucleosides that differs from any one of SEQ ID NOs: 562-761, 772-781, or 798-800 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 19 consecutive nucleosides.

[0158] In certain embodiments, the second strand comprises a nucleoside sequence of at least 21 consecutive nucleosides that differs from any one of SEQ ID NOs: 562-761, 772-781, or 798-800 by 0 or 1 nucleoside, and the second strand has a region that is at least 85% complementary to the first strand over 21 consecutive nucleosides.

[0159] In certain embodiments, the second strand comprises any one of SEQ ID NOs: 562-761, 772-781, or 798-800.

[0160] The modification patterns of the nucleic acids shown in Sequence Nos. 562 to 761, 772 to 781, or 798 to 800 are summarized in Table 4 below.

[0161]

Table 5

[0162] As used herein, particularly in Tables 3 and 4, the following abbreviations are used for modified nucleosides.

[0163] 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.

[0164] Furthermore, the letter "s" is used as an abbreviation for a phosphorothioate linkage between two consecutive (modified) nucleosides. For example, the abbreviation "AmsAm" is used for two consecutive 2'-O-methyl-adenosine nucleosides linked via a 3'5' phosphorothioate linkage. No abbreviation is used for nucleosides linked via a standard 3'5' phosphodiester linkage. For example, the abbreviation "AmAm" is used for two consecutive 2'-O-methyl-adenosine nucleosides linked via a 3'5' phosphodiester linkage.

[0165] In certain embodiments, the nucleic acid comprises, consists of, or consists essentially of a first strand having a (modified) nucleoside sequence that differs from any one of SEQ ID NOs: 362-561, 762-771, 782-786, or 795-797 by 0 or 1 nucleoside, and a second strand comprising, consisting of, or consisting essentially of a (modified) nucleoside sequence that differs from any one of SEQ ID NOs: 562-761, 772-781 or 798-800 by 0 or 1 nucleoside.

[0166] Preferred combinations of complementary modified antisense (first) and sense (second) strands are listed in Table 5 below.

[0167] [Table 6] JPEG2025519224000033.jpg244152JPEG2025519224000034.jpg244149JPEG2025519224000035.jpg246151

[0168] In particularly preferred embodiments, the invention relates to a nucleic acid comprising, consisting of, or consisting essentially of a first strand and a second strand having a nucleoside sequence that differs from any one of the following first and second sequences by 0 or 1 nucleoside.

[0169] [Table 7]

[0170] In even more preferred embodiments, the invention relates to a nucleic acid comprising, consisting of, or consisting essentially of a first strand and a second strand having a nucleoside sequence that differs from any one of the following first and second sequences by 0 or 1 nucleoside.

[0171] [Table 8]

[0172] If there is any ambiguity between the sequences in this specification and the sequences in the attached sequence listing, the sequences provided in this specification shall be regarded as the correct sequences.

[0173] Apurinic / apyrimidinic nucleotide In certain embodiments, the nucleic acids according to the invention contain one, for example two, for example three, for example four, or more apurinic / apyrimidinic nucleosides. Apurinic / apyrimidinic nucleosides are modified nucleosides because they lack the base normally found at the 1-position of the sugar moiety. Typically, a hydrogen will be present at the 1-position of the sugar moiety of the apurinic / apyrimidinic nucleosides present in the nucleic acids according to the invention.

[0174] Apurinic / apyrimidinic nucleosides are present in the terminal region of the second strand and are preferably located within the terminal 5 nucleosides at the end of the strand. The terminal region may be the terminal 5 nucleosides including the apurinic / apyrimidinic nucleoside.

[0175] The second strand may include the following preferred features (all combinations are particularly contemplated unless mutually exclusive): Two or more apurinic / apyrimidinic nucleosides in the terminal region of the second strand, and / or Two or more apurinic / apyrimidinic nucleosides in either the 5' or 3' terminal region of the second strand, and / or Two or more apurinic / apyrimidinic nucleosides in either the 5' or 3' terminal region of the second strand, which are present in overhangs as described herein, and / or Two or more consecutive apurinic / apyrimidinic nucleosides in the terminal region of the second strand, preferably with one such apurinic / apyrimidinic nucleoside being the terminal nucleoside, two or more consecutive apurinic / apyrimidinic nucleosides, and / or Two or more consecutive abasic nucleosides in either the 5' or 3' terminal region of the second strand, preferably one such abasic nucleoside is the terminal nucleoside at either the 5' or 3' terminal region of the second strand, two or more consecutive abasic nucleosides, and / or Reverse internucleoside linkages connecting at least one abasic nucleoside to adjacent nucleoside bases in the terminal region of the second strand, and / or Reverse internucleoside linkages connecting at least one abasic nucleoside to adjacent nucleoside bases in either the 5' or 3' terminal region of the second strand, and / or An abasic nucleoside as the penultimate nucleoside connected via a reverse linkage to a nucleoside that is not the terminal nucleoside (referred to herein as the third last nucleoside), and / or Abasic nucleosides as two terminal nucleosides connected via a 5'-3' linkage when reading the strand in the direction towards the end containing the terminal nucleoside, Abasic nucleosides as two terminal nucleosides connected via a 3'-5' linkage when reading the strand in the direction towards the end containing the terminal nucleoside, Abasic nucleosides at the last two positions of the end, where the penultimate nucleoside is connected to the third last nucleoside via a reverse linkage, and the reverse linkage is a 5-5' reverse linkage or a 3'-3' reverse linkage, Abasic nucleosides at the last two positions of the end, where the penultimate nucleoside is connected to the third last nucleoside via a reverse linkage, (1) The reverse linkage is a 5-5' reverse linkage, and the linkage between the end and the penultimate abasic nucleoside is 3'5' when reading towards the end containing the end and the penultimate abasic nucleoside, or (2) The reverse linkage is a 3-3’ reverse linkage, and the linkage between the terminal and the second last abasic nucleoside is either 5’3’ when read towards the terminal including the terminal and the second last abasic nucleoside, the abasic nucleoside.

[0176] Preferably, an abasic nucleoside is present at the end of the second strand.

[0177] Preferably, in the terminal region of the second strand, preferably at the terminal and the second last position, two or at least two abasic nucleosides are present.

[0178] Preferably, two or more abasic nucleosides are consecutive, for example, all abasic nucleosides may be consecutive. For example, the terminal 1 nucleotide or the terminal 2 nucleotides or the terminal 3 nucleotides or the terminal 4 nucleotides may be abasic nucleosides.

[0179] Also, except when only one abasic nucleoside is present at the end, the abasic nucleoside may be linked to the adjacent nucleoside via a 5’-3’ phosphodiester linkage or a reverse linkage. When only one abasic nucleoside is present, it will have a reverse linkage with the adjacent nucleoside.

[0180] The reverse linkage (sometimes called an inverted linkage, which is also seen in the art) includes any of the 5’-5’, 3-’3’, 3’-2’, or 2’-3’ phosphodiester linkages between adjacent sugar moieties of nucleosides.

[0181] Abasic nucleosides that are not at the end will each have two phosphodiester linkages, one with each adjacent nucleoside, and they may be reverse linkages or 5’-3 phosphodiester bonds or one of each.

[0182] The preferred embodiment includes two abasic nucleosides at the end of the second strand and at the second position from the end, and the inverse internucleoside linkage is located between the second (abasic) nucleoside from the end and the third nucleoside from the end.

[0183] Preferably, two abasic nucleosides are present at the end of the second strand and at the second position from the end, the second nucleoside from the end is linked to the third nucleoside from the end via an inverse internucleoside linkage, and is linked to the terminal nucleoside via a 5'-3' or 3'-5' phosphodiester linkage (when read in the direction of the end of the molecule).

[0184] Various preferred features are as follows. The inverse internucleoside linkage is a 3'-3' inverse linkage. The inverse internucleoside linkage is present in the terminal region distal from the 5'-terminal phosphate of the second strand.

[0185] The inverse internucleoside linkage is a 5'-5' inverse linkage. The inverse internucleoside linkage is present in the terminal region distal from the 3'-terminal hydroxide of the second strand.

[0186] In certain embodiments, the second strand comprises two consecutive abasic nucleosides in the 5' terminal region of the second strand, one such abasic nucleoside being the terminal nucleoside of the 5' terminal region of the second strand and the other abasic nucleoside being the second last nucleoside of the 5' terminal region of the second strand, (a) said second last abasic nucleoside is linked to the adjacent first base nucleoside of the adjacent 5' proximal region via a reverse internucleoside linkage, (b) the reverse linkage is a 5-5' reverse linkage, and (c) the linkage between the terminal and the second last abasic nucleoside is 3'5' when read towards the terminus containing the terminal and the second last abasic nucleoside. More typically, (i) the first and second strands each have a length of 23 nucleosides, (ii) two phosphorothioate internucleoside linkages are present between three consecutive positions in said 5' proximal region of the second strand, the first phosphorothioate internucleoside linkage is present between (a) said adjacent first base nucleoside and the adjacent second base nucleoside of said 5' proximal region of the second strand, the second phosphorothioate internucleoside linkage is present between said adjacent second base nucleoside and the adjacent third base nucleoside of said 5' proximal region of the second strand, (iii) two phosphorothioate internucleoside linkages are present between three consecutive positions in both the 5' and 3' terminal regions of the first strand, the terminal nucleosides in each of said 5' and 3' terminal regions of the first strand are each attached to the adjacent second last nucleosides at 5' and 3' respectively by a phosphorothioate internucleoside linkage, and each of the first 5' and 3' second last nucleosides is attached to the adjacent third last nucleosides at 5' and 3' respectively by a phosphorothioate internucleoside linkage, and (iv) the second strand of the nucleic acid is conjugated directly or indirectly to one or more ligand moieties in the 3' terminal region of the second strand.

[0187] Alternatively, the second strand preferably contains two consecutive abasic nucleosides in the overhang of the 3'-terminal region of the second strand, one such abasic nucleoside being the terminal nucleoside of the 3'-terminal region of the second strand and the other abasic nucleoside being the second last nucleoside of the 3'-terminal region of the second strand, (a) said second last abasic nucleoside being connected via an inverse internucleoside linkage to the adjacent first base nucleoside of the adjacent 3'-proximal region, (b) the inverse linkage being a 3-3' inverse linkage, and (c) the linkage between the terminal and the second last abasic nucleoside being 5'-3' when read towards the terminus containing the terminal and the second last abasic nucleoside. More typically, (i) the first and second strands each have a length of 23 nucleosides, (ii) two phosphorothioate internucleoside linkages are present between three consecutive positions of said 3'-proximal region of the second strand, the first phosphorothioate internucleoside linkage being present between (a) said adjacent first base nucleoside and the adjacent second base nucleoside of said 3'-proximal region of the second strand, and the second phosphorothioate internucleoside linkage being present between said adjacent second base nucleoside and the adjacent third base nucleoside of said 3'-proximal region of the second strand, (iii) two phosphorothioate internucleoside linkages are present between three consecutive positions of both the 5'- and 3'-terminal regions of the first strand, the terminal nucleosides in each of said 5'- and 3'-terminal regions of the first strand each being attached via a phosphorothioate internucleoside linkage to the adjacent second last nucleoside at 5' and 3', respectively, and each of the first 5'- and 3'-second last nucleosides being attached via a phosphorothioate internucleoside linkage to the adjacent third last nucleoside at 5' and 3', respectively, and (iv) the second strand of the nucleic acid is directly or indirectly conjugated with one or more ligand moieties in the 5'-terminal region of the second strand.

[0188] Examples of such structures are as follows (the specific RNA nucleosides shown are not limiting and may be any RNA nucleoside). A 3'-3' inverse linkage (and the last phosphodiester bond between two abasic molecules in the 5'-3' direction when read towards the ends of the molecule is also shown)

[0189]

Chemical formula

[0190]

Chemical formula

[0191] One or more abasic nucleosides present in the nucleic acid are provided in the presence of one or more inverse nucleoside linkages, i.e., 5'-5' or 3'-3' inverse nucleoside linkages. The inverse linkage results from a change in the orientation of the adjacent nucleoside sugars, such that the sugars will have a 3'-5' orientation as opposed to the conventional 5'-3' orientation (based on the numbering of the ring atoms of the nucleoside sugar). One or more abasic nucleosides present in the nucleic acids of the invention preferably contain such inverted nucleoside sugars.

[0192] When the terminal nucleoside has an inverted orientation, this will result in an "inversion" of the terminal configuration of the entire nucleic acid. Certain structures illustrated and referred to herein are represented using the conventional 5'-3' direction (based on the numbering of the ring atoms of the nucleoside sugar), but the change in orientation and the presence of a terminal nucleoside with a proximal 3'-3' inverse linkage will result in a nucleic acid having an overall 5'-5' terminal structure (i.e., the conventional 3' terminal nucleoside becomes the 5' terminal nucleoside), which will be understood. Alternatively, it will be understood that the change in orientation and the presence of a terminal nucleoside with a proximal 5'-5' inverse linkage will result in a nucleic acid having an overall 3'-3' terminal structure.

[0193] The proximal 3'-3' or 5'-5' reverse linkages described herein may include a reverse linkage that is directly adjacent / attached to a terminal nucleoside having reverse orientation, e.g., a single terminal nucleoside having reverse orientation. Alternatively, the proximal 3'-3' or 5'-5' reverse linkages described herein may include a reverse linkage that is adjacent to two or more nucleosides having reverse orientation, e.g., two or more terminal region nucleosides having reverse orientation such as the terminal and the penultimate nucleoside. Thus, the reverse linkage may be attached to the penultimate nucleoside having reverse orientation. One of ordinary skill in the art will understand that the reverse orientation described above can result in a nucleic acid molecule having an overall 3'-3' or 5'-5' end structure as described herein, but that if one or more additional reverse linkages and / or nucleosides having reverse orientation are present, the overall nucleic acid can also have a 3'-5' end structure corresponding to the 5' / 3' ends of the conventional configuration.

[0194] In one aspect, the nucleic acid may have a 3'-3' reverse linkage, and the terminal sugar moiety may contain a 5' OH rather than a 5' phosphate group at the 5' position of its terminal sugar.

[0195] Thus, one of ordinary skill in the art will clearly understand that the 5'-5', 3'-3', and 3'-5' (read in the direction of its terminus) end variants of the more general 5'-3' structure (based on the numbering of the ring atoms of the terminal nucleoside sugar) depicted herein are within the scope of the present disclosure if one or more reverse linkages are present.

[0196] For example, in the context of one or more nucleosides having an inverse orientation that creates inverse internucleoside linkages and / or inverse termini, where the relative position of the linkage (e.g., relative to a linker) or the position of internal features (e.g., modified nucleosides) is defined relative to the 5' or 3' end of the nucleic acid, the 5' or 3' end is the conventional 5' or 3' end that would have been present if the inverse linkage were not placed, and the conventional 5' or 3' end is determined by considering the orientation of the majority of the internal nucleoside linkages and / or nucleoside orientations within the nucleic acid. From such internal linkages and / or nucleoside orientations, it is possible to determine which end of the nucleic acid constitutes the conventional 5' and 3' ends (based on the numbering of the ring atoms of the terminal nucleoside sugar) of a molecule in the absence of the inverse linkage.

[0197] For example, in the structure shown below, abasic residues are present at the first two positions located at the "5'" end. When the terminal nucleoside has an inverse orientation, the "5'" end, which is the conventional 5' end shown in the figure below, can actually contain a 3'OH in light of the inverted nucleoside at the terminal position. Nevertheless, when read in the standard 5'[PO4] to 3'[OH] direction of the nucleic acid molecule (based on the numbering of the ring atoms of the nucleoside sugar), most of the molecule will contain conventional internucleoside linkages that extend from the 3'OH of the sugar to the 5' phosphate of the next sugar, and this can be used to determine the conventional 5' and 3' ends where the absence of an inverted terminus configuration is found. A 5’A-A-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me3’

[0198] The inverse linkage is preferably located at the end of a nucleic acid, e.g., RNA, distal from the ligand portion of the molecule, e.g., the GalNAc-containing portion.

[0199] A GalNAc-siRNA construct having 5'-GalNAc on the sense strand can have an inverse linkage at the end opposite the sense strand.

[0200] A GalNAc-siRNA construct having 3'-GalNAc on the sense strand can have an inverted linkage at the opposite end of the sense strand.

[0201] Length of nucleic acid In one embodiment, i) the first strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides, and / or ii) the second strand of the nucleic acid has a length in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0202] Typically, the double-stranded region of the nucleic acid is 17 to 30 nucleosides in length, more preferably 19 or 21 nucleosides in length. Similarly, the complementary region between the first strand and the portion of the RNA transcribed from the ZPI gene is 17 to 30 nucleosides in length. Generally, the double-stranded structure of a nucleic acid, such as an iRNA, is about 15 to 30 base pairs in length, such as 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length. Ranges and lengths intermediate to those described above are also contemplated as part of the present invention.

[0203] Similarly, the region of complementarity of the antisense sequence to the target sequence and / or the region of complementarity of the antisense sequence to the sense sequence is about 15 to 30 nucleosides in length, such as 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleosides in length. Ranges and lengths intermediate to those described above are also contemplated as part of the present invention.

[0204] In certain preferred embodiments, the region of complementarity of the antisense sequence to the target sequence and / or the region of complementarity of the antisense sequence to the sense sequence is at least 17 nucleosides in length. For example, the region of complementarity between the antisense strand and the target is 19 to 21 nucleosides in length, for example, the region of complementarity is 21 nucleosides in length.

[0205] In preferred embodiments, each strand is 30 nucleosides or less in length.

[0206] In certain preferred embodiments, the double-stranded structure of the nucleic acid, such as siRNA, is 19 or 21 base pairs in length. In particularly preferred embodiments, the double strand may have one of the following structures. For example, ETXM316 - ETXM415, ETXM436 - ETXM515, and ETXM1064 - ETXM1198:

[0207]

Chemical Formula

[0208]

Chem.

[0209] The nucleic acids described in this specification, such as dsRNA, may further contain one or more single-stranded nucleoside overhangs, such as 1 to 4, 2 to 4, 1 to 3, 2 to 3, 1, 2, 3, or 4 nucleosides. The nucleoside overhangs may contain or consist of nucleoside / nucleoside analogs including deoxynucleosides / nucleosides. The overhangs may be present in the sense strand, the antisense strand, or any combination thereof. Further, the nucleosides of the overhang may be present at the 5'-end, 3'-end, or both ends of the antisense strand or sense strand of the nucleic acid, such as dsRNA.

[0210] In certain preferred embodiments, at least one strand contains a 3'-overhang of at least one nucleoside, for example, at least one strand contains a 3'-overhang of at least two nucleosides. The overhangs are preferably present in the antisense / guide strand and / or the sense / passenger strand.

[0211] Nucleic acid modification In certain embodiments, the nucleic acids of the present invention, such as RNA, such as dsiRNA, do not include further modifications, such as chemical modifications or conjugations known in the art and described herein.

[0212] In other preferred embodiments, the nucleic acids of the present invention, such as RNA, such as dsiRNA, are further chemically modified to enhance stability or other beneficial properties.

[0213] In certain embodiments of the present invention, substantially all nucleosides are modified.

[0214] The nucleic acids characterized by the present invention can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA. This document is hereby incorporated herein by reference.

[0215] Modifications include terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverse ligation) or 3'-terminal modifications (conjugation, DNA nucleosides in RNA, or RNA nucleosides in DNA, inverse ligation, etc.); base modifications, such as stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, substitution by conjugate bases; sugar modifications (e.g., at the 2'- or 4'-position) or sugar substitution; or backbone modifications, including modifications or substitutions of the phosphodiester linkage, such as those that include a modified backbone or do not include a native internucleoside linkage.

[0216] Specific examples of nucleic acids, such as siRNA compounds useful in the embodiments described herein, include, but are not limited to, RNAs that include a modified backbone or do not include a native internucleoside linkage. Nucleic acids such as RNAs with a modified backbone include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification and as sometimes referred to in the art, modified nucleic acids that do not have a phosphorus atom in the internucleoside backbone, such as RNA, can also be considered oligonucleosides. In some embodiments, modified nucleic acids, such as siRNA, will have a phosphorus atom in their internucleoside backbone.

[0217] Modified nucleic acids, such as for the RNA backbone, include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3'-alkylene phosphonates and chiral phosphonates including methyl and other alkyl phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and the normal 3'-5' linkages, boranophosphates having their 2'-5' linkage analogs, and those having an inverted polarity where adjacent pairs of nucleoside units are linked 5'-3' or 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0218] Also, modified nucleic acids, such as RNA, may contain one or more substituted sugar moieties. Nucleic acids characterized herein, such as siRNA, such as dsiRNA, may contain at the 2'-position one of the following: OH, F, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl may or may not be substituted. 2'-O-methyl and 2'-F are preferred modifications.

[0219] In certain preferred embodiments, the nucleic acid contains at least one modified nucleoside.

[0220] The nucleic acids of the invention may contain one or more modified nucleosides in the first strand and / or the second strand.

[0221] In some embodiments, substantially all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand contain modifications.

[0222] In some embodiments, all of the nucleosides of the sense strand and substantially all of the nucleosides of the antisense strand contain modifications.

[0223] In some embodiments, all of the nucleosides of the sense strand and all of the nucleosides of the antisense strand include modifications.

[0224] In one embodiment, at least one of the modified nucleosides is selected from the group consisting of: deoxy-nucleosides, 3'-terminal deoxy-thymidine (dT) nucleosides, 2'-O-methyl modified nucleosides (also referred to herein as 2'-Me, where Me is methoxy), 2'-fluoro modified nucleosides, 2'-deoxy modified nucleosides, locked nucleosides, unlocked nucleosides, nucleosides with restricted conformation, constrained ethyl nucleosides, abasic nucleosides, 2'-amino modified nucleosides, 2'-O-allyl modified nucleosides, 2'-C-alkyl modified nucleosides, 2'-hydroxy modified nucleosides, 2'-methoxyethyl modified nucleosides, 2'-O-alkyl modified nucleosides, morpholino nucleosides, phosphoramidates, nucleosides containing unnatural bases, tetrahydropyran modified nucleosides, 1,5-anhydrohexitol modified nucleosides, cyclohexenyl modified nucleosides, nucleosides containing phosphorothioate groups, nucleosides containing methylphosphonate groups, nucleosides containing 5'-phosphate, and nucleosides containing 5'-phosphate mimics. In another embodiment, the modified nucleoside includes a short sequence of 3'-terminal deoxy-thymidine nucleoside (dT).

[0225] The modification of the nucleoside can preferably be selected from the group including, but not limited to, LNA, HNA, CeNA, 2-methoxyethyl, 2'-O-alkyl, 2-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In another embodiment, the modification of the nucleoside is a 2'-O-methyl ("2-Me") or 2'-fluoro modification.

[0226] One preferred modification is a modification selected, optionally, from a 2'-Me modification or a 2'-F modification at the 2'-OH group of ribose sugar.

[0227] Preferred nucleic acids contain one or more nucleosides in the first strand and / or the second strand, which are modified as follows to form modified nucleosides.

[0228] A nucleic acid, wherein the modification is a modification selected, optionally, from a 2'-Me modification or a 2'-F modification at the 2'-OH group of ribose sugar.

[0229] A nucleic acid, wherein the first strand contains a 2'-F modification at the 2nd, 6th, 14th position, or any combination thereof, counted from the 1st position of the first strand.

[0230] A nucleic acid, wherein the second strand contains a 2'-F modification at the 7th, 9th, 11th position, or any combination thereof, counted from the 1st position of the second strand.

[0231] A nucleic acid, wherein the second strand contains a 2'-F modification at the 7th and / or 9th and / or 11th and / or 13th position, counted from the 1st position of the second strand.

[0232] A nucleic acid, wherein the second strand contains a 2'-F modification at the 7th, 9th, and 11th position, counted from the 1st position of the second strand.

[0233] A nucleic acid, wherein the first strand and the second strand each contain a 2'-Me modification and a 2'-F modification.

[0234] A nucleic acid containing at least one heat destabilizing modification, preferably at one or more of the 1st to 9th positions of the first strand, counted from the 1st position of the first strand, and / or at one or more of the positions of the second strand aligned with the 1st to 9th positions of the first strand, wherein the destabilizing modification is selected from modified unlocked nucleic acid (UNA) and glycol nucleic acid (GNA), preferably glycol nucleic acid.

[0235] A nucleic acid having three or more 2'-F modifications at positions 7 to 13 of the second strand, counted from position 1 of the second strand, for example, having 4, 5, 6, or 7 2'-F modifications at positions 7 to 13 of the second strand.

[0236] The second strand is a nucleic acid having at least three, for example, 4, 5, or 6 2'-Me modifications at positions 1 to 6 of the second strand, counted from position 1 of the second strand.

[0237] The first strand is a nucleic acid having at least five consecutive 2'-Me modifications in the 3'-terminal region, preferably including the terminal nucleoside of the 3'-terminal region, or within at least 1 or 2 nucleosides from the terminal nucleoside of the 3'-terminal region.

[0238] The first strand is a nucleic acid having seven consecutive 2'-Me modifications in the 3'-terminal region, preferably including the terminal nucleoside of the 3'-terminal region.

[0239] A nucleic acid having at least one heat destabilizing modification at position 7 of the first strand, counted from position 1 of the first strand.

[0240] A nucleic acid that is an siRNA oligonucleotide, wherein the siRNA oligonucleotide has at least three 2'-F modifications at positions 6 to 12 of the second strand, counted from position 1 of the second strand.

[0241] A nucleic acid that is an siRNA oligonucleotide, wherein the second strand has at least three 2'-Me modifications at positions 1 to 6 of the second strand, counted from position 1 of the second strand.

[0242] A nucleic acid that is an siRNA oligonucleoside, wherein each of the first strand and the second strand comprises an alternating modification pattern, preferably a fully alternating modification pattern, along the entire length of each of the first strand and the second strand, and the nucleosides of the first strand are modified by (i) 2’Me modification of the odd-numbered nucleosides counted from the 1-position of the first strand, and (ii) 2’F modification of the even-numbered nucleosides counted from the 1-position of the first strand, and the nucleosides of the second strand are modified by (i) 2’F modification of the odd-numbered nucleosides counted from the 1-position of the second strand, and (ii) 2’Me modification of the even-numbered nucleosides counted from the 1-position of the second strand. Typically, such a fully alternating modification pattern is present in blunt-ended oligonucleosides, and each of the first strand and the second strand is 19 or 23 nucleosides in length.

[0243] The 1-position of the first strand or the second strand is closest to the end of the nucleic acid (any abasic nucleosides are ignored), and is the nucleoside joined to the adjacent nucleoside (at the 2-position) via an internal 3’ to 5’ bond when read in the direction away from that end of the molecule, with reference to the bond between the sugar moieties of the backbone.

[0244] Thus, it can be understood that the "1-position of the sense strand" is the 5'-most nucleoside of the conventional 5'-end of the sense strand (excluding abasic nucleosides). Typically, this 1-position nucleoside of the sense strand will be equivalent to the 5'-nucleoside of the selected target nucleic acid sequence. More generally, the sense strand will have nucleosides equivalent to those of the target nucleic acid sequence starting from this 1-position of the sense strand, although acceptable mismatches between the sequences are also possible.

[0245] As used herein, the "1-position of the antisense strand" is the 5'-most nucleoside of the conventional 5'-end of the antisense strand (excluding abasic nucleosides). As described above, there will be a region of complementarity between the sense strand and the antisense strand, and thus the antisense strand will also have a region of complementarity to the target nucleic acid sequence referred to above.

[0246] In certain embodiments, the nucleic acid, e.g., an RNAi agent, further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage. For example, the phosphorothioate or methylphosphonate internucleoside linkage may be present at the 3' end or terminal region of one strand, i.e., the sense or antisense strand, or at the termini of both strands, the sense and antisense strands.

[0247] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkage may be present at the 5' end or terminal region of one strand, i.e., the sense or antisense strand, or at the termini of both strands, the sense and antisense strands.

[0248] In certain embodiments, the phosphorothioate or methylphosphonate internucleoside linkage may be present at both the 5' and 3' ends or terminal regions of one strand, i.e., the sense or antisense strand, or at the termini of both strands, the sense and antisense strands.

[0249] Any of the nucleic acids may optionally contain one or more phosphorothioate (PS) modifications within the nucleic acid, e.g., at least two PS internucleoside linkages at the termini of the strand.

[0250] At least one of the oligoribonucleoside strands preferably contains at least two consecutive phosphorothioate modifications at the last three nucleosides of the oligonucleotide.

[0251] Accordingly, the invention also relates to a nucleic acid as disclosed herein, which comprises phosphorothioate internucleoside linkages between at least two or three consecutive positions, such as the 5' and / or 3' terminal regions and / or near-terminal regions of the second strand, wherein the near-terminal region preferably abuts the terminal region where one or more abasic nucleosides of the second strand are located.

[0252] The nucleic acids disclosed herein each contain phosphorothioate internucleoside linkages between at least two or three consecutive positions in the 5' and / or 3' terminal regions of the first strand, and preferably, the terminal positions in the 5' and / or 3' terminal regions of the first strand are attached to their adjacent positions by phosphorothioate internucleoside linkages.

[0253] The nucleic acid strand may be an RNA containing phosphorothioate internucleoside linkages between three consecutive nucleosides contiguous to two abasic nucleosides located at the termini.

[0254] Preferred nucleic acids are double-stranded RNAs containing two adjacent abasic nucleosides at the 5' end of the second strand and a ligand moiety containing one or more GalNAc ligand moieties at the 3' end on the opposite side of the second strand. More preferably, the same nucleic acid may further contain phosphorothioate linkages between the nucleotides at positions 3-4 and 4-5 of the second strand when read from position 1 of the second strand. Even more preferably, the same nucleic acid may further contain 2'-F modifications at positions 7, 9, and 11 of the second strand.

[0255] The following structure:

[0256]

Chemical formula

[0257] The modified nucleosides of the second strand have a modification pattern (5'-3') by any one of the following: Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me、 comprising a nucleic acid, wherein (s) is a phosphorothioate internucleoside linkage.

[0258] The modified nucleosides of the second strand have a modification pattern (5'-3') by any one of the following: ia-ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or ia-ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, comprising, wherein ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in the overhang of two nucleosides, a nucleic acid.

[0259] The modified nucleoside of the second strand has a modification pattern (5'-3') by any one of the following: ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, or Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, or Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, comprising wherein (s) is a phosphorothioate nucleoside internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3'-end of the second strand, the inverted abasic nucleoside is present in a 2-nucleoside overhang, a nucleic acid.

[0260] The modified nucleosides have the following modification patterns: Modification pattern 1: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me or Modification pattern 2: Second strand (5'-3'): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me or Modification pattern 3: Second strand (5'-3'): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 6: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, A nucleic acid containing any one of them.

[0261] The modified nucleoside has the following modified patterns: Modified pattern 1: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 2: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 3: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 4: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 5: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 6: Second strand (5’-3’): Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, including any one of them, where (s) is a phosphorothioate internucleoside linkage, a nucleic acid.

[0262] The modified nucleoside has the following modified pattern: Modified Pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me or Modified Pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me or Modified Pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me or Modified Pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me or Modified Pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 6: 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, including any one of them, Wherein (s) is a phosphorothioate internucleoside linkage, a nucleic acid.

[0263] The modified nucleoside has the following modified patterns: Modified pattern 1: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, first strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 2: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 3: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5'-3'): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 4: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 5: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 6: Containing any one of the second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, first strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Wherein ia represents an inverted abasic nucleoside, a nucleic acid.

[0264] The modified nucleoside has the following modified patterns: Modified pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me-ia-ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, first strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, First strand (5’-3’): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me Or modified pattern 6: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me-ia-ia, - First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, including any one of them Wherein, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is present in the overhang of two nucleosides, nucleic acid.

[0265] The modified nucleoside has the following modified pattern: Modified pattern 1: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 2: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 3: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 4: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 5: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 6: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, including any one of them, wherein, (s) is a phosphorothioate nucleoside internucleoside linkage, and ia represents an inverted abasic nucleoside, a nucleic acid.

[0266] The modified nucleoside has the following modification patterns: Modification pattern 1: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-F-F-F-F-Me-Me-Me-Me-Me-Me-Me-F(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 2: Second strand (5’-3’): Me-Me-Me-Me-Me-F-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 3: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modification pattern 4: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-F-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 5: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me Or modified pattern 6: Second strand (5’-3’): Me-Me-Me-Me-Me-Me-F-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me(s)Me(s)Me-ia-ia, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, comprising any one of them, wherein (s) is a phosphorothioate nucleoside internucleoside linkage, ia represents an inverted abasic nucleoside, and when the inverted abasic nucleoside represented by ia-ia is present at the 3’ end of the second strand, the inverted abasic nucleoside is present in a 2-nucleoside overhang, nucleic acid.

[0267] The first strand contains a 2’ sugar modification pattern, the modification is selected from at least 2’Me and 2’F sugar modifications, provided that the total number of 2’F sugar modifications in the first strand does not consist of 4 2’F modifications nor 6 2’F modifications, nucleic acid.

[0268] The first strand contains a 2’ sugar modification pattern, the modification is selected from at least 2’Me and 2’F sugar modifications, and the total number of 2’F sugar modifications in the first strand consists of 3, 5, or 7 2’F modifications, nucleic acid.

[0269] The first strand contains a 2’ sugar modification pattern, the modification is selected from at least 2’Me and 2’F sugar modifications, and the total number of 2’F sugar modifications in the first strand consists of 3 2’F modifications, nucleic acid.

[0270] The first strand contains a 2'-sugar modification pattern, said modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications of the first strand consists of 5 2'-F modifications, a nucleic acid.

[0271] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 including wherein X2, X3, and X4 are selected from 2'-Me and 2'-F sugar modifications, provided that at least one of X2, X3, and X4 is a 2'-F sugar modification and the other two sugar modifications are 2'-Me sugar modifications, a nucleic acid.

[0272] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 including wherein X2 is a 2'-F sugar modification and X3 and X4 are 2'-Me sugar modifications, a nucleic acid.

[0273] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 including wherein X3 is a 2'-F sugar modification and X2 and X4 are 2'-Me sugar modifications, a nucleic acid.

[0274] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-(Me)7-(F-Me)2-X3-Me-X4-(Me)3 including wherein X4 is a 2'-F sugar modification and X2 and X3 are 2'-Me sugar modifications, a nucleic acid.

[0275] The first strand contains a 2'-sugar modification pattern, said modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 7 2'-F modifications, a nucleic acid.

[0276] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 and wherein X2, X3, and X4 are selected from 2'-Me and 2'-F sugar modifications, provided that at least one of X2, X3, and X4 is a 2'-F sugar modification and the other two sugar modifications are 2'-Me sugar modifications, a nucleic acid.

[0277] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 and wherein X2 is a 2'-F sugar modification and X3 and X4 are 2'-Me sugar modifications, a nucleic acid.

[0278] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 and wherein X3 is a 2'-F sugar modification and X2 and X4 are 2'-Me sugar modifications, a nucleic acid.

[0279] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-Me-X2-Me-F-Me-(F)2-(Me)4-(F-Me)2-X3-Me-X4-(Me)3 and wherein X4 is a 2'-F sugar modification and X2 and X3 are 2'-Me sugar modifications, a nucleic acid.

[0280] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7 comprising a nucleic acid, wherein X1 is a thermolabile modification.

[0281] The first strand has the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7 comprising a nucleic acid, wherein X1 is a thermolabile modification.

[0282] The second strand has the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 a nucleic acid comprising

[0283] The second strand has the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 , comprising a nucleic acid, wherein the first strand comprises a 2'-sugar modification pattern selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand does not consist of four 2'-F modifications nor six 2'-F modifications.

[0284] The second strand has the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 comprising a nucleic acid, wherein the first strand comprises a 2'-sugar modification pattern selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of three, five, or seven 2'-F modifications.

[0285] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, wherein X1 is a thermolabile modification, a nucleic acid.

[0286] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): (Me-F)3-(Me)7-F-Me-F-(Me)7 and comprising, a nucleic acid.

[0287] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 , and comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5 A nucleic acid comprising

[0288] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 , comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3 A nucleic acid comprising

[0289] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 , comprising the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7, wherein X1 is a thermolabile modification, a nucleic acid.

[0290] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand having the following 2'-sugar modification pattern (5'-3'): (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6 a nucleic acid.

[0291] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand having the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5 a nucleic acid.

[0292] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification pattern (5'-3'): (Me)8-(F)3-(Me) 10 and comprising the nucleosides of the first strand having the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3 a nucleic acid.

[0293] The second strand has the following 2'-sugar and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising a nucleic acid, wherein ia represents an inverted abasic nucleoside.

[0294] The second strand has the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The first strand comprises a 2'-sugar modification pattern, said modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand does not consist of 4 2'-F modifications nor 6 2'-F modifications, a nucleic acid.

[0295] The second strand has the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The first strand comprises a 2'-sugar modification pattern, said modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of 3, 5, or 7 2'-F modifications, a nucleic acid.

[0296] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-X1-(Me)7-F-Me-F-(Me)7, wherein X1 is a thermolabile modification, a nucleic acid.

[0297] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): (Me-F)3-(Me)7-F-Me-F-(Me)7 A nucleic acid comprising the same.

[0298] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-(Me)7-(F-Me)2-F-(Me)5 A nucleic acid comprising the same.

[0299] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand are the following 2'-sugar modification patterns (5'-3'): Me-F-(Me)3-F-(Me)7-F-Me-F-(Me)3-F-(Me)3 A nucleic acid comprising.

[0300] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand are the following 2'-sugar modification and abasic modification patterns (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand are the following 2'-sugar modification patterns (5'-3'): Me-F-(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)7, comprising, wherein X1 is a thermolabile modification, a nucleic acid.

[0301] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand are the following 2'-sugar modification and abasic modification patterns (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 comprising, wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand are the following 2'-sugar modification patterns (5'-3'): (Me-F)3-Me-(F)2-(Me)4-(F-Me)2-(Me)6 A nucleic acid comprising.

[0302] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)5 A nucleic acid comprising the same.

[0303] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-(Me)8-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me-F-(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-(Me)3 A nucleic acid comprising the same.

[0304] The second strand has the following 2'-sugar modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein, ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, a nucleic acid.

[0305] The second strand has the following 2'-sugar modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The first strand contains a 2'-sugar modification pattern, said modification being selected from at least 2'-Me and 2'-F sugar modifications, provided that the total number of 2'-F sugar modifications in the first strand is neither four 2'-F modifications nor six 2'-F modifications, nucleic acid.

[0306] The second strand has the following 2'-sugar modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The first strand contains a 2'-sugar modification pattern, said modification being selected from at least 2'-Me and 2'-F sugar modifications, and the total number of 2'-F sugar modifications in the first strand consists of three, five, or seven 2'-F modifications, nucleic acid.

[0307] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-X1-(Me)7-F-Me-F-(Me)5(s)Me(s)Me, wherein X1 is a thermally destabilizing modification, nucleic acid.

[0308] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)Me-F-Me-F-(Me)7-F-Me-F-(Me)5(s)Me(s)Me A nucleic acid comprising the same.

[0309] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-(Me)7-(F-Me)2-F-(Me)3(s)Me(s)Me A nucleic acid comprising the same.

[0310] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-(Me)7-F-Me-F-(Me)3-F-Me(s)Me(s)Me A nucleic acid comprising the above.

[0311] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, the nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-X1-Me-(F)2-(Me)4-F-Me-F-(Me)5(s)Me(s)Me, wherein X1 is a thermally destabilizing modification, A nucleic acid.

[0312] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)Me-F-Me-F-Me-(F)2-(Me)4-(F-Me)2-(Me)4(s)Me(s)Me A nucleic acid comprising the same.

[0313] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-F-(Me)3(s)Me(s)Me A nucleic acid comprising the same.

[0314] A nucleic acid comprising a first strand that is at least partially complementary to a portion of RNA transcribed from a target gene, and a second strand that is at least partially complementary to the first strand, wherein the first strand and the second strand form a double-stranded region that is at least 17 nucleosides in length, and the nucleosides of the second strand have the following 2'-sugar modification and abasic modification pattern (5'-3'): ia-ia-Me(s)Me(s)(Me)6-(F)3-(Me) 10 wherein ia represents an inverted abasic nucleoside and (s) represents a phosphorothioate linkage, The nucleosides of the first strand have the following 2'-sugar modification pattern (5'-3'): Me(s)F(s)(Me)3-F-Me-(F)2-(Me)4-(F-Me)2-(Me)2-F-Me(s)Me(s)Me A nucleic acid comprising the above.

[0315] Preferred modifications are as follows. Modification pattern 1: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me-F-Me-Me-Me-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, where X1 is a thermolabile modification; Or modification pattern 2: Second strand (5'-3'): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5'-3'): Me-F-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me ; Or modification pattern 3: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me ; Or modification pattern 4: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-Me ; Or modification pattern 5: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me, where X1 is a thermolabile modification; Or modification pattern 6: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me ; Or modification pattern 7: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-F-F-Me- Me-Me-Me-F-Me-F-Me-F-Me-Me-Me-Me-Me ; or modification pattern 8: Second strand (5’-3’): ia-ia-Me-Me-Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me-F-Me-Me-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me-Me-Me

[0316] Particularly preferred modifications are as follows. Modification pattern 1: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-X1-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where X1 is a thermolabile modification; or modification pattern 2: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me ; or modification pattern 3: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me; or modified pattern 4: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-Me-Me-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me ; or modified pattern 5: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-X1-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me, where X1 is a thermally labile modification; or modified pattern 6: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-F-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-Me-Me(s)Me(s)Me ; or modified pattern 7: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-F-F-Me - Me-Me-Me-F-Me-F-Me-F-Me-Me-Me(s)Me(s)Me ; or modified pattern 8: Second strand (5’-3’): ia-ia-Me(s)Me(s)Me-Me-Me-Me-Me-Me-F-F-F-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me, First strand (5’-3’): Me(s)F(s)Me-Me-Me-F-Me-F-F-Me-Me-Me-Me-F-Me-F-Me-Me-Me-F-Me(s)Me(s)Me; wherein (s) represents a phosphorothioate internucleoside linkage.

[0317] Conjugation of Nucleic Acids with Ligands Another modification of the nucleic acids of the invention, such as RNA, such as siRNA, involves conjugating the nucleic acid, such as siRNA, to one or more ligand moieties to enhance, for example, the activity, cellular distribution, or cellular uptake into cells of the nucleic acid, such as siRNA.

[0318] In some embodiments, the described ligand moieties may be attached to a nucleic acid, such as a siRNA oligonucleoside, via a linker that may or may not be cleavable. The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, such as covalently attaching two parts of a compound.

[0319] The ligand can be attached to the 3’ or 5’ end of the sense strand.

[0320] The ligand is preferably conjugated to the 3’ end of the sense strand of a nucleic acid, such as a siRNA agent.

[0321] Accordingly, in a further aspect, the invention relates to a conjugate for inhibiting the expression of a target, such as a target gene, in a cell, said conjugate comprising a nucleic acid moiety and one or more ligand moieties, wherein said nucleic acid moiety comprises a nucleic acid disclosed herein.

[0322] In one aspect, the second strand of the nucleic acid is conjugated directly or indirectly (e.g., via a linker) to one or more ligand moieties, and the ligand moieties are typically present in the terminal region of the second strand, preferably in its 3' terminal region.

[0323] In certain embodiments, the ligand moiety comprises GalNAc or a GalNAc derivative attached to the nucleic acid, e.g., dsiRNA, via a linker.

[0324] Accordingly, the present invention relates to conjugates comprising a ligand moiety that is i) one or more GalNAc ligands, and / or ii) one or more GalNAc ligand derivatives, and / or iii) one or more GalNAc ligands conjugated to the nucleic acid via a linker .

[0325] The GalNAc ligand may be conjugated directly or indirectly to the 5' or 3' terminal region of the second strand of the nucleic acid, preferably to its 3' terminal region.

[0326] GalNAc ligands are well known in the art and are described, inter alia, in European Patent No. 3775207.

[0327] In some embodiments, the ligand moiety comprises one or more ligands.

[0328] In some embodiments, the ligand moiety comprises one or more carbohydrate ligands.

[0329] In some embodiments, the one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and / or polysaccharides.

[0330] In some embodiments, the one or more carbohydrates include one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0331] In some embodiments, the one or more carbohydrates include one or more N-acetyl-galactosamine moieties.

[0332] In some embodiments, the compounds described anywhere herein include two or three N-acetylgalactosamine moieties.

[0333] In some embodiments, one or more ligands are attached in a linear configuration or a branched configuration, for example, each configuration is attached to a branch point of the entire linker.

[0334] Exemplary linear and exemplary branched configurations are shown in FIGS. 1a and 1b.

[0335] In FIG. 1a (linear), (a) and / or (b) can typically represent a connecting bond or a connecting group such as a phosphate group or a phosphorothioate group.

[0336] In FIG. 1b (branched), in some embodiments, one or more ligands are attached as a bifurcated or trifurcated branched configuration. Typically, a trifurcated branched configuration such as an N-acetylgalactosamine trifurcated branched configuration may be preferred.

[0337] Linker Exemplary compounds of the present invention include a "linker portion" that is part of all "linkers", such as those illustrated in formula (I).

[0338]

Chemical formula

[0339] As will be further understood in the art, exemplary compounds of the invention include all linkers positioned between the oligonucleoside moiety and the ligand moiety of such compounds. The total linker thereby "connects" the oligonucleoside moiety and the ligand moiety to each other.

[0340] The linker is often conceptually envisioned to include one or more linker building blocks. For example, there is a linker portion illustrated as the "linker portion" presented in formula (I), which is disposed adjacent to the ligand portion and typically attaches the ligand portion directly or indirectly to the oligonucleoside portion via a branching point. The linker portion illustrated in formula (I) is often also referred to as the "ligand arm or group of arms" of the linker. There may further be an additional linker portion between the oligonucleoside portion and the branching point, but it does not always exist. The additional linker portion is often referred to as the "tether portion" of the linker that "tethers" the oligonucleoside portion to the remainder of the conjugate compound. Such "ligand arms" and / or "linker portions" and / or "tether portions" can be envisioned by referring to the linear and / or branched chain configurations shown above.

[0341] As can be understood from the claims and the remainder of this patent specification, the scope of the invention extends to linear or branched chain configurations and there is no limitation on the number of individual ligands that may be present. Further, the reader will also recognize that there are numerous structures that can be used as linker portions based on the state of the art and the expertise of oligonucleoside chemists.

[0342] The remainder of the linker (other than the linker portion) shown in the claims and the remainder of the patent specification is represented by its chemical composition in formula (I) which the inventors consider to be particularly unique to the present invention. However, more generally speaking, such chemical compositions can be described as the "tether portion" as described above, and the "tether portion" is the portion of the linker that includes the group of atoms between Z, i.e., the oligonucleoside portion and the linker portion, as illustrated in formula (I).

[0343] The tether portion of formula I Regarding formula (I), the "tether portion" includes the atomic group between Z, i.e., the oligonucleoside portion, and the linker portion.

[0344] In some embodiments, R1 is hydrogen at each occurrence. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl.

[0345] In some embodiments, R2 is hydroxy. In some embodiments, R2 is halo. In some embodiments, R2 is fluoro. In some embodiments, R2 is chloro. In some embodiments, R2 is bromo. In some embodiments, R2 is iodo. In some embodiments, R2 is nitro.

[0346] In some embodiments, X1 is methylene. In some embodiments, X1 is oxygen. In some embodiments, X1 is sulfur.

[0347] In some embodiments, X2 is methylene. In some embodiments, X2 is oxygen. In some embodiments, X2 is sulfur.

[0348] In some embodiments, m = 3.

[0349] In some embodiments, n = 6.

[0350] In some embodiments, X1 is oxygen and X2 is methylene. In some embodiments, both X1 and X2 are methylene.

[0351] 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.

[0352] 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.

[0353] Thus, in some embodiments, exemplary compounds of the present invention include the following structure.

[0354]

Chemical formula

[0355] 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.

[0356] Thus, in some embodiments, exemplary compounds of the present invention include the following structure.

[0357]

Chemical formula

[0358] Alternative tether moiety During the synthesis of the compounds of the present invention, alternative tether moiety structures may occur. In some embodiments, the alternative tether moiety has a change in one or more atoms of the tether moiety of the entire linker as compared to the tether moiety described anywhere in this specification.

[0359] In some embodiments, the alternative tether moiety is a compound of formula (I) described anywhere in this specification, wherein R2 is hydroxy.

[0360] 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.

[0361] Thus, in some embodiments, the compounds of the present invention include the following structure.

[0362]

Chem.

[0363] 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.

[0364] Thus, in some embodiments, the compounds of the present invention include the following structure.

[0365]

Chem.

[0366] Linker moiety With respect to formula (I), the "linker moiety" shown in formula (I) includes a group of atoms located between the tether moiety described anywhere in this specification and the ligand moiety described anywhere in this specification.

[0367] In some embodiments, as shown in formula (I) described anywhere in this specification

[0368]

Chem.

[0369]

Chem.

[0370] [Chemical formula] In the formula, A I is hydrogen or a suitable hydroxy protecting group, a is an integer of 2 or 3, c and d are independently an integer of 1 to 6, or

[0371] [Chemical formula] In the formula, A I is hydrogen or a suitable hydroxy protecting group, a is an integer of 2 or 3, e is an integer of 2 to 10.

[0372] In some embodiments, the part shown in formula (I):

[0373] [Chemical formula] is formula (VIa),

[0374] [Chemical formula] In the formula, A I is hydrogen or a suitable hydroxy protecting group, a is 3, b is an integer of 3.

[0375] In some embodiments, the part shown in formula (I) described anywhere in this specification:

[0376] [Chemical formula] is formula (VII),

[0377] [Chemical formula] In the formula, A I is hydrogen, a is an integer of 2 or 3, preferably 3.

[0378] Another exemplary compound of the present invention includes a "linker portion" that is part of all "linkers" illustrated in formula (I * ), and

[0379] [Chemical formula] In the formula, r and s are independently integers selected from 1 to 16, Z is an oligonucleoside moiety.

[0380] As will be further understood in the art, exemplary compounds of the present invention include all linkers located between the oligonucleoside moiety and the ligand moiety of such compounds. The all linkers thereby "link" the oligonucleoside moiety and the ligand moiety to each other.

[0381] The all linkers are often conceptually envisioned to include one or more linker building blocks. For example, there is a linker portion illustrated as the "linker portion" presented in formula (I * ), and this linker portion is disposed adjacent to the ligand portion and typically attaches the ligand portion directly or indirectly to the oligonucleoside portion via a branching point. Formula (I *) The linker portion shown in is often also referred to as the "ligand arm or group of arms" of the entire linker. There may be an additional linker portion between the oligonucleoside portion and the branching point, but it does not always exist. The additional linker portion is often referred to as the "tether portion" of the entire linker, which "tethers" the oligonucleoside portion to the remainder of the conjugate compound. Such "ligand arms" and / or "linker portions" and / or "tether portions" can be envisioned by referring to the linear and / or branched chain configurations shown above.

[0382] As can be understood from the claims and the remainder of this patent specification, the scope of the present invention extends to linear or branched chain configurations and there is no limitation on the number of individual ligands that may be present. Furthermore, the reader will also recognize that there are numerous structures that can be used as linker portions based on the state of the art and the expertise of oligonucleoside chemists.

[0383] The remainder of the entire linker (other than the linker portion) shown in the claims and the remainder of the patent specification is represented by its chemical composition in formula (I), which the inventors consider to be particularly unique to the present invention. However, more generally speaking, such chemical compositions can be described as the "tether portion" as described above, and the "tether portion" is the portion of the entire linker that includes the group of atoms between Z, i.e., the oligonucleoside portion and the linker portion, as shown in formula (I).

[0384] Tether portion Formula (I * ) With respect to, the "tether portion" includes the group of atoms between Z, i.e., the oligonucleoside portion and the linker portion.

[0385] In some embodiments, s is an integer selected from 4 to 12. In some embodiments, s is 6.

[0386] In some embodiments, r is an integer selected from 4 to 14. In some embodiments, r is 6. In some embodiments, r is 12.

[0387] In some embodiments, r is 12 and s is 6.

[0388] Thus, in some embodiments, exemplary compounds of the invention include the following structure.

[0389]

Chemical formula

[0390] In some embodiments, r is 6 and s is 6.

[0391] Thus, in some embodiments, exemplary compounds of the invention include the following structure.

[0392]

Chemical formula

[0393] Linker moiety With respect to formula (I * ), the "linker moiety" illustrated in formula (I) includes a group of atoms located between the tether moiety described anywhere in this specification and the ligand moiety described anywhere in this specification.

[0394] In some embodiments, the moiety illustrated in formula (I * ) described anywhere in this specification:

[0395]

Chemical formula

[0396]

Chem.

[0397]

Chem.

[0398]

Chem.

[0399] In some embodiments, the part shown in formula (I):

[0400]

Chem.

[0401]

Chem.

[0402] In some embodiments, the portion illustrated in formula (I) described anywhere in this specification:

[0403]

Chemical formula

[0404]

Chemical formula

[0405] In some embodiments, a = 2. In some embodiments, a = 3. In some embodiments, b = 3.

[0406] Vectors and cells In one aspect, the present invention provides a cell containing a nucleic acid such as an inhibitory RNA [RNAi] described herein.

[0407] In one aspect, the present invention provides a cell containing a vector described herein.

[0408] In one aspect, the present invention provides a vector containing an oligonucleotide inhibitor, such as iRNA, such as siRNA.

[0409] Pharmaceutically acceptable compositions In one aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of a target gene, and the composition contains an inhibitor such as an oligomer such as a nucleic acid disclosed herein.

[0410] The pharmaceutically acceptable composition may contain an excipient and / or a carrier.

[0411] Some examples of substances that can serve as pharmaceutically acceptable carriers include the following: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0412] Typical pharmaceutical carriers include, but are not limited to, the following: binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose, etc.), fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or calcium hydrogen phosphate, etc.), lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (such as starch, sodium starch glycolate, etc.), and wetting agents (such as sodium lauryl sulfate, etc.).

[0413] For formulating the composition of the present invention, pharmaceutically acceptable organic or inorganic excipients that do not cause adverse reactions with nucleic acids and are suitable for parenteral administration can also be used. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone, etc.

[0414] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oil bases. The solution may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not cause adverse reactions with nucleic acids and are suitable for parenteral administration can be used.

[0415] In one embodiment, the nucleic acid or composition is administered in a non-buffered solution. In certain embodiments, the non-buffered solution is saline or water. In other embodiments, the nucleic acid, such as an RNAi agent, is administered in a buffered solution. In such embodiments, the buffer solution may contain acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffer solution may be phosphate buffered saline (PBS).

[0416] Dosage The pharmaceutical composition of the present invention can be administered in a dosage sufficient to inhibit gene expression or modify the expression or function of a target. Generally, when the composition contains a nucleic acid, a suitable dosage of the nucleic acid of the present invention, such as siRNA, is in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, and generally will be in the range of about 1 to 50 mg per kilogram of body weight per day. Typically, a suitable dosage of the nucleic acid of the present invention, such as siRNA, will be in the range of about 0.1 mg / kg to about 5.0 mg / kg, such as about 0.3 mg / kg to about 3.0 mg / kg.

[0417] A repeated dosage regimen may include administering a therapeutic amount of a nucleic acid, such as siRNA, periodically, such as once a day or once a year. In certain embodiments, the nucleic acid, such as siRNA, is administered about once a month to once every three months (i.e., once every three months).

[0418] In various embodiments, the nucleic acid, such as an siRNA agent, is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the nucleic acid, such as an siRNA agent, is administered at a dose of about 10 mg / kg to about 30 mg / kg. In certain embodiments, the nucleic acid, such as an siRNA agent, is administered at a dose selected from about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg. In certain embodiments, the nucleic acid, such as an siRNA agent, is administered at a dose of about 0.1 mg / kg to about 5.0 mg / kg once a week, once a month, once every two months, or once every quarter (i.e., once every three months). In certain embodiments, the nucleic acid, such as an siRNA agent, is administered to the subject once a week. In certain embodiments, the nucleic acid, such as an siRNA agent, is administered to the subject once a month. In certain embodiments, the nucleic acid, such as an siRNA agent, is administered about once every quarter (i.e., about once every three months).

[0419] After the initial treatment regimen, treatment may be administered less frequently. For example, after administering once a week or once every two weeks over a three-month period, the administration may be repeated once a month, once every six months, or once a year or longer.

[0420] The pharmaceutical composition can be administered once a day, or as two, three, or more divided doses at appropriate intervals throughout the day, or even using delivery by sustained infusion or controlled release formulations. In that case, the nucleic acid, such as siRNA, contained in each divided dose must be made proportionally less in order to achieve the total daily dosage. Dosage units can also be formulated to be delivered over several days, for example, using conventional sustained release formulations that provide for the sustained release of the nucleic acid, such as siRNA, over several days. Sustained release formulations are well known in the art and are particularly useful for the delivery of agents at specific sites, such as those that can be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0421] In other embodiments, a single dose of the pharmaceutical composition may have long-term persistence, such that subsequent doses are administered at intervals within 3, 4, or 5 days, or at intervals within 1, 2, 3, or 4 weeks. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once every two months. In certain embodiments, the siRNA is administered about once a month to about once every quarter (i.e., about once every three months), or even once every six months or once every twelve months.

[0422] The estimation of the effective dosage and in vivo half-life of an individual nucleic acid, such as siRNA, encompassed by the present invention can be carried out using conventional methodologies or based on in vivo tests using appropriate animal models known in the art.

[0423] The pharmaceutical composition of the present invention can be administered in a number of ways depending on whether local or systemic treatment is desired and on the site to be treated. Administration can be local (e.g., by transdermal patch), pulmonary, e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer, intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subcutaneous, e.g., by an implantable device, or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular administration. In certain preferred embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

[0424] In one embodiment, a nucleic acid, such as an siRNA agent, is administered subcutaneously to a subject.

[0425] An inhibitor, such as a nucleic acid, such as siRNA, can be delivered to target a specific tissue (e.g., specific hepatocytes).

[0426] Methods for inhibiting gene expression, or methods for inhibiting target expression or function The present invention also provides methods for inhibiting gene expression in cells, as well as methods for inhibiting the expression and / or function of other target molecules. Such methods include contacting a cell with an effective amount of a nucleic acid of the present invention, such as an siRNA agent, such as a double-stranded siRNA, that is effective for inhibiting gene expression in the cell, thereby inhibiting gene expression in the cell. In a preferred embodiment, the gene is ZPI.

[0427] Contact of the cell with an inhibitor, such as a nucleic acid, such as an siRNA, such as a double-stranded siRNA agent, can be carried out in vitro or in vivo. Contacting the cell with an inhibitor nucleic acid, such as an siRNA, in vivo includes contacting a cell or cell population within a subject, such as a human subject, with the nucleic acid, such as an siRNA. Combinations of in vitro and in vivo methods for contacting the cell are also possible. As discussed above, the contact with the cell may be direct or indirect. Further, the contact with the cell can be achieved by a target-directed ligand moiety that includes any ligand moiety described herein or known in the art. In a preferred embodiment, the target-directed ligand moiety is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand moiety that directs the siRNA agent to the site of interest.

[0428] The term "inhibit", as used herein, is used interchangeably with "reduce", "silence", "down-regulate", "suppress", and other similar terms, and includes any level of inhibition.

[0429] In some embodiments of the methods of the invention, the expression or activity of the gene or inhibitory target is preferably inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% as determined by qPCR as described herein and / or when siRNA is introduced into target cells by transfection, or inhibited to a level below the detection level of the assay. In certain embodiments, such methods include clinically relevant inhibition of the expression of the target gene, as demonstrated, for example, by clinically relevant outcomes after treating a subject with an agent that reduces gene expression and / or target activity.

[0430] In some embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the ZPI gene with an IC50 value lower than 2500 pM, 2400 pM, 2300 pM, 2200 pM, 2100 pM, 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM, preferably as determined by qPCR as described herein, more preferably by reverse transcriptase (RT)-qPCR.

[0431] In preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the ZPI gene with an IC50 value lower than 2500 pM. In more preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the ZPI gene with an IC50 value lower than 1000 pM. In even more preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the ZPI gene with an IC50 value lower than 500 pM. In the most preferred embodiments, when the nucleic acids of the invention are transfected into cells, they inhibit the expression of the ZPI gene with an IC50 value lower than 100 pM.

[0432] Inhibition of ZPI gene expression can be quantified by the following method.

[0433] Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB cell bank) may be maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37°C in a 5% CO2 atmosphere. Next, siRNA duplex 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)) may be transfected into the cells using 10×3-fold serial dilutions over a final duplex concentration range of 20 nM to 1 pM. Transfection can be carried out by adding 9.7 μL of Opti-MEM (ThermoFisher) + 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture may be incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells may be incubated at 37°C / 5% CO2 for 24 hours before purifying total RNA using the RNeasy 96 kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in a single experiment.

[0434] cDNA synthesis can be carried out using the FastQuant RT (with gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be carried out using the FastStart Universal Probe Master kit (Roche) with primers specific for human ZPI (Hs01547819_m1) and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or ABI QuantStudio7.

[0435] qPCR can be performed in duplicate on cDNA derived from each well to calculate the average cycle threshold (Ct). The comparative Ct (ΔΔCt) method can be used to calculate relative ZPI expression from the average Ct values and normalize to GAPDH and to untreated cells. The maximum percent inhibition and IC50 values of ZPI expression can be calculated using a four-parameter (variable slope) model using GraphPad Prism9.

[0436] Alternatively or additionally, inhibition of ZPI gene expression can be characterized by a reduction in the average relative expression of the ZPI gene.

[0437] In some embodiments, when cells are transfected with 0.1 nM of the nucleic acid of the invention, the average relative expression of ZPI, as measured herein, preferably by qPCR, more preferably by reverse transcriptase (RT)-qPCR, is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4.

[0438] In some embodiments, when cells are transfected with 5 nM of the nucleic acid of the invention, the average relative expression of ZPI, as measured herein, preferably by qPCR, more preferably by reverse transcriptase (RT)-qPCR, is below 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3.

[0439] The average relative expression of the ZPI gene can be quantified by the following method.

[0440] Huh7 cells (a human hepatocyte-derived cell line, obtained from the JCRB cell bank) may be maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere. An siRNA duplex targeting ZPI mRNA or a negative control siRNA (siRNA control, sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 794), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 790)) may be transfected into the cells at final duplex concentrations of 5 nM and 0.1 nM. Transfection can be carried out by adding 9.7 μL of Opti-MEM (ThermoFisher) + 0.3 μL of Lipofectamine RNAiMAX (ThermoFisher) to 10 μL of each siRNA duplex. The mixture may be incubated at room temperature for 15 minutes and then added to 100 μL of complete growth medium containing 20,000 Huh7 cells. The cells may be incubated at 37 °C / 5% CO2 for 24 hours before purifying total RNA using the RNeasy 96 kit (Qiagen). Each duplex can be tested by transfection in duplicate wells in two independent experiments.

[0441] cDNA synthesis can be carried out using the FastQuant RT (with gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be carried out using the FastStart Universal Probe Master kit (Roche) with primers specific for human ZPI (Hs01547819_m1) and human GAPDH (Hs02786624_g1) on an ABI Prism 7900HT or an ABI QuantStudio 7.

[0442] qPCR can be carried out in duplicate on the cDNA derived from each well and the average Ct can be calculated. Relative ZPI expression can be calculated from the average Ct values using the comparative Ct (ΔΔCt) method and normalized to GAPDH and to untreated cells.

[0443] Inhibition of gene expression may be represented by a reduction in the amount of mRNA of the target gene of interest compared to a suitable control. Inhibition of target function may be represented by a reduction in the activity of the target compared to a suitable control.

[0444] In other embodiments, inhibition of gene or other target expression may be evaluated in terms of a reduction in gene expression, such as protein expression or a parameter functionally related to a signaling pathway.

[0445] Method for treating or preventing a disease associated with the expression of a target gene expression / function The present invention also provides a method for reducing or inhibiting gene expression in a cell or for reducing target expression or function using a nucleic acid of the present invention, such as siRNA, or a composition comprising a nucleic acid of the present invention, such as siRNA. Such methods include contacting the cell with a nucleic acid of the present invention, such as dsiRNA, and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the gene, thereby inhibiting gene expression in the cell. Reduction of target gene expression or function can be evaluated by any method known in the art. In a preferred embodiment, the gene is ZPI.

[0446] In the method of the present invention, the cell may be contacted in vitro or in vivo, i.e., the cell may be present within a subject.

[0447] A cell suitable for treatment using the method of the present invention may be any cell that expresses the target gene or target of interest associated with the disease.

[0448] The in vivo method of the present invention involves administering to a subject a composition comprising a nucleic acid of the present invention, such as siRNA, wherein the nucleic acid, such as iRNA, comprises a nucleoside sequence complementary to at least a portion of the RNA transcript of a gene of a mammal to be treated, or to another nucleic acid whose expression and / or function is associated with a disease.

[0449] The present invention further provides a method for treating a subject in need thereof. The treatment method of the present invention involves administering to a subject, for example, a nucleic acid such as siRNA of the present invention, or a nucleic acid such as siRNA targeting a gene, or a pharmaceutical composition comprising a nucleic acid targeting a gene, in a therapeutically effective amount to a subject who would benefit from a reduction or inhibition of gene expression and / or expression and / or function of a target.

[0450] The nucleic acid of the present invention, such as siRNA, may be administered as a "naked" nucleic acid or "naked" siRNA administered in the absence of a pharmaceutical composition. The naked nucleic acid may be present in a suitable buffer solution. The buffer solution may contain acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution can be adjusted to be suitable for administration to a subject.

[0451] Alternatively, the nucleic acid of the present invention, such as siRNA, may be administered as a pharmaceutical composition such as a dsiRNA liposome formulation.

[0452] In one embodiment, the method involves administering the composition described herein such that the expression of the target gene decreases over, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the expression of the target gene decreases for a long period, for example, at least about 2, 3, 4 days or longer, for example, about 1 week, 2 weeks, 3 weeks, or 4 weeks, or longer, for example, about 1 month, 2 months, or 3 months.

[0453] The subject can be administered a therapeutic amount of a nucleic acid, such as from about 0.01 mg / kg to about 200 mg / kg, such as siRNA.

[0454] The nucleic acid, such as siRNA, can be administered by intravenous infusion over a regular period of time. In certain embodiments, after an initial treatment regimen, treatment may be administered at a lower frequency. Administration of siRNA can reduce, for example, the gene product level of a target gene in a patient's cells or tissues to at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the detection level of the assay used. In certain embodiments, administration results in clinical stabilization or preferably a clinically relevant reduction of at least one sign or symptom of the gene-related disorder.

[0455] Alternatively, the nucleic acid, such as siRNA, can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver the desired daily dose of the nucleic acid, such as siRNA, to the subject. The injections may be repeated over a period of time. Administration may be repeated regularly. In certain embodiments, after an initial treatment regimen, treatment may be administered at a lower frequency. A repeated dose regimen may include administering a therapeutic amount of the nucleic acid regularly, such as once a day to once a year. In certain embodiments, the nucleic acid is administered about once a month to about once every quarter (i.e., once every about 3 months).

[0456] In one aspect, the present invention can be applied to the compounds, methods, compositions, or uses of Proposition Numbers 1 to 101 below (references to any of the formulas of Propositions 1 to 101 refer only to the formulas defined within Propositions 1 to 101. Such formulas are reproduced in Figure 6).

[0457] 1. The following structure:

[0458]

Chemical formula

[0459] 2. The compound according to claim 1, wherein R1 is hydrogen at each occurrence.

[0460] 3. The compound according to claim 1, wherein R1 is methyl.

[0461] 4. The compound according to claim 1, wherein R1 is ethyl.

[0462] 5. The compound according to any one of claims 1 to 4, wherein R2 is hydroxy.

[0463] 6. The compound according to any one of claims 1 to 4, wherein R2 is halo.

[0464] 7. The compound according to claim 6, wherein R2 is fluoro.

[0465] 8. The compound according to claim 6, wherein R2 is chloro.

[0466] 9. The compound according to Proposition 6, wherein R2 is bromo.

[0467] 10. The compound according to Proposition 6, wherein R2 is iodo.

[0468] 11. The compound according to Proposition 6, wherein R2 is nitro.

[0469] 12. The compound according to any one of Propositions 1 to 11, wherein X1 is methylene.

[0470] 13. The compound according to any one of Propositions 1 to 11, wherein X1 is oxygen.

[0471] 14. The compound according to any one of Propositions 1 to 11, wherein X1 is sulfur.

[0472] 15. The compound according to any one of Propositions 1 to 14, wherein X2 is methylene.

[0473] 16. The compound according to any one of Propositions 1 to 15, wherein X2 is oxygen.

[0474] 17. The compound according to any one of Propositions 1 to 16, wherein X2 is sulfur.

[0475] 18. The compound according to any one of Propositions 1 to 17, wherein m = 3.

[0476] 19. The compound according to any one of Propositions 1 to 18, wherein n = 6.

[0477] 20. X1 is oxygen, X2 is methylene, preferably, q = 1, r = 2, s = 1, t = 1, v = 1, the compound according to Propositions 13 and 15.

[0478] 21. Both X1 and X2 are methylene, preferably, q = 1, r = 3, s = 1, t = 1, v = 1, the compounds described in Propositions 12 and 15.

[0479] 22. Z is,

[0480]

Chemical formula

[0481] 23. The oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene, the compound described in Proposition 22.

[0482] 24. The RNA compound comprises an RNA duplex comprising a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, and each of the first strand and the second strand has 5' and 3' ends, the compound described in Proposition 23.

[0483] 25. The RNA compound has an adjacent phosphate attached at the 5' end of its second strand, the compound described in Proposition 24.

[0484] 26. The RNA compound has an adjacent phosphate attached at the 3' end of its second strand, the compound described in Proposition 24.

[0485] 27. The compound of formula (II).

[0486] [Chemistry]

[0487] 28. The compound of formula (III).

[0488] [Chemistry]

[0489] 29. The oligonucleoside comprises an RNA duplex including a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand, and is the compound according to Proposition 27 or 28.

[0490] 30. A composition comprising the compound of formula (II) defined in Proposition 27 and the compound of formula (III) defined in Proposition 28, and optionally dependent on Proposition 29.

[0491] 31. The composition according to Proposition 30, wherein the compound of formula (III) defined in Proposition 28 is present in an amount in the range of 10 to 15% by weight of the composition.

[0492] 32. The compound of formula (IV).

[0493] [Chemistry]

[0494] 33. The compound of formula (V).

[0495] [Chemistry]

[0496] 34. The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, the first strand being at least partially complementary to the RNA sequence of the target gene, the second strand being at least partially complementary to the first strand, each of the first strand and the second strand having 5' and 3' ends, and the RNA duplex being attached to an adjacent phosphate at the 3' end of its second strand, a compound according to claim 32 or 33.

[0497] 35. A composition comprising a compound of formula (IV) as defined in claim 32 and a compound of formula (V) as defined in claim 33, optionally dependent on claim 34.

[0498] 36. The composition according to claim 35, wherein the compound of formula (V) as defined in claim 33 is present in an amount in the range of 10 to 15% by weight of the composition.

[0499] 37. The compound according to any one of claims 1 to 29 or 32 to 34, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0500] 38. The modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy, the compound according to claim 37.

[0501] 39. The compound according to any one of claims 1 to 29, 32 to 34, or 37 to 38, wherein the oligonucleoside further comprises one or more degradation protection moieties at one or more termini.

[0502] 40. The one or more degradation protection moieties are not present at the termini of the oligonucleoside strand carrying the ligand moiety, and / or the one or more degradation protection moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleosides are present at the distal end of the strand carrying the ligand moiety, the compound according to claim 39.

[0503] 41. The ligand moiety shown in formula (I) of Proposition 1 is a compound according to any one of Propositions 1 to 29, 32 to 34, or 37 to 40, comprising one or more ligands.

[0504] 42. The ligand moiety shown in formula (I) of Proposition 1 is a compound according to Proposition 41, comprising one or more carbohydrate ligands.

[0505] 43. The one or more carbohydrates of the compound according to Proposition 42 may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[0506] 44. The one or more carbohydrates of the compound according to Proposition 43 comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0507] 45. The one or more carbohydrates of the compound according to Proposition 44 comprise one or more N-acetyl-galactosamine moieties.

[0508] 46. The compound according to Proposition 45 comprises two or three N-acetylgalactosamine moieties.

[0509] 47. The one or more ligands of the compound according to any one of Propositions 41 to 46 are attached in a linear or branched configuration.

[0510] 48. The one or more ligands of the compound according to Proposition 47 are attached as a bifurcated or trifurcated branched configuration.

[0511] 49. The moiety shown in formula (I) of Proposition 1:

[0512]

Chemical formula

[0513]

Chemical formula

[0514]

Chemical formula

[0515]

Chemical formula

[0516] 50. The said part illustrated in formula (I) of Proposition 1:

[0517]

Chemical formula

[0518] [Chemical] wherein, A I is hydrogen, a is an integer of 2 or 3, the compound according to Propositions 46 to 48.

[0519] 51. The compound according to Proposition 49 or 50, wherein a = 2.

[0520] 52. The compound according to Proposition 49 or 50, wherein a = 3.

[0521] 53. The compound according to Proposition 49, wherein b = 3.

[0522] 54. The compound of formula (VIII).

[0523] [Chemical]

[0524] 55. The compound of formula (IX).

[0525] [Chemical]

[0526] 56. The oligonucleoside comprises an RNA duplex including a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' termini, and the RNA duplex is attached to an adjacent phosphate at the 5' terminus of its second strand, the compound according to Proposition 54 or 55.

[0527] 57. A composition comprising the compound of formula (VIII) defined in Proposition 54 and the compound of formula (IX) defined in Proposition 55, optionally dependent on Proposition 56.

[0528] 58. The composition according to Proposition 57, wherein the compound of formula (IX) defined in Proposition 55 is present in an amount in the range of 10 to 15% by weight of the composition.

[0529] 59. A compound of formula (X).

[0530]

Chemical formula

[0531] 60. A compound of formula (XI).

[0532]

Chemical formula

[0533] 61. An oligonucleoside comprising an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to an RNA sequence of a target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand, the compound according to Proposition 59 or 60.

[0534] 62. A composition comprising a compound of formula (X) defined in Proposition 59 and a compound of formula (XI) defined in Proposition 60, optionally dependent on Proposition 61.

[0535] 63. The composition according to Proposition 62, wherein the compound of formula (XI) defined in Proposition 60 is present in an amount in the range of 10 to 15% by weight of the composition.

[0536] 64. The compound according to any one of Propositions 54 to 63, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0537] 65. The modification is a compound according to claim 64 selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0538] 66. The oligonucleoside is a compound according to any one of claims 54 to 65 further comprising one or more deprotecting moieties at one or more termini.

[0539] 67. The one or more deprotecting moieties are not present at the termini of the oligonucleoside chain carrying the ligand moiety and / or the one or more deprotecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, the inverted abasic nucleosides being present at the distal end of the chain carrying the ligand moiety as shown in any one of formulas (VIII), (IX), (X), or (XI) of claims 54, 55, 59, or 60, a compound according to claim 66.

[0540] 68. A method for preparing a compound according to any one of claims 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of claims 30, 31, 35, 36, 57, 58, 62, 63, comprising formulas (XII) and (XIII):

[0541]

Chemical formula

[0542] 69. The compound of formula (XII) is the compounds of formula (XIV) and (XV):

[0543]

Chemical formula

[0544] 70. The compound of formula (XII) is of formula (XIIa),

[0545]

Chemical formula

[0546] [Chem.] The oligonucleoside comprises an RNA duplex containing a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand. A method according to claim 68 for 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.

[0547] 71. The compound of formula (XII) is of formula (XIIb),

[0548] [Chem.] The compound of formula (XIII) is of formula (XIIIa),

[0549] [Chem.] The oligonucleoside comprises an RNA duplex containing a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand. The method according to Proposition 68 for preparing a compound according to any of Propositions 20, 25, 28, 29, 55, 56 and / or a composition according to any of Propositions 30, 31, 57, 58.

[0550] 72. The compound of formula (XII) is of formula (XIIc),

[0551] [Chemical formula] The compound of formula (XIII) is of formula (XIIIa),

[0552] [Chemical formula] The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' ends, and the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand. The method according to Proposition 68 for preparing a compound according to any of Propositions 21, 26, 32, 34, 59, 61 and / or a composition according to any of Propositions 35, 36, 62, 63.

[0553] 73. The compound of formula (XII) is of formula (XIId),

[0554] [Chemical formula] The compound of formula (XIII) is of formula (XIIIa),

[0555] [Chemical formula] The oligonucleoside comprises an RNA duplex containing a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' termini, and the RNA duplex is attached to an adjacent phosphate at the 3' terminus of its second strand. The method according to claim 68 for 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.

[0556] 74. The compound of formula (XIIIa) is of formula (XIIIb):

[0557] [Chemical formula] The method according to any one of claims 70 to 73, which is as follows.

[0558] 75. The compound of formula (XIV) is either of formula (XIVa) or formula (XIVb),

[0559] [Chemical formula] The compound of formula (XV) is either of formula (XVa) or formula (XIVb),

[0560] [Chemical formula] The oligonucleoside comprises an RNA duplex including a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' ends, (i) for the RNA duplex of formula (XV a), at the 5' end of its second strand, it is attached to an adjacent phosphate, or (ii) for the RNA duplex of formula (XV b), at the 3' end of its second strand, it is attached to an adjacent phosphate. The method according to claim 69, which is dependent on claims 70 to 73.

[0561] 76. A compound of formula (XII),

[0562]

Chemical formula

[0563] 77. A compound of formula (XIIa).

[0564]

Chemical formula

[0565] 78. A compound of formula (XIIb).

[0566] [ka]

[0567] 79. A compound of formula (XIIc).

[0568] [ka]

[0569] 80. A compound of formula (XIId).

[0570] [ka]

[0571] 81. A compound of formula (XIII):

[0572] [ka] During the ceremony, R1, in each occurrence, is independently selected from the group consisting of hydrogen, methyl, and ethyl; m is an integer from 1 to 6; n is an integer from 1 to 10; compound.

[0573] 82. A compound of formula (XIIIa).

[0574] [ka]

[0575] 83. A compound of formula (XIIIb).

[0576] [ka]

[0577] 84. A compound of formula (XIV),

[0578] [Chemical formula] wherein, R1 is selected from the group consisting of hydrogen, methyl, and ethyl, R2 is selected from the group consisting of hydrogen, hydroxy, -OC 1~3 alkyl, -C(=O)OC 1~3 alkyl, halo, and nitro, X2 is selected from the group consisting of methylene, oxygen, and sulfur, s, t, v are independently integers from 0 to 4, provided that s, t, and v cannot all be 0 simultaneously, a compound.

[0579] 85. A compound of formula (XIVa).

[0580] [Chemical formula]

[0581] 86. A compound of formula (XIVb).

[0582] [Chemical formula]

[0583] 87. A compound of formula (XV),

[0584] [Chemical formula] wherein, R1 is independently selected from the group consisting of hydrogen, methyl, and ethyl at each occurrence, X1 is selected from the group consisting of methylene, oxygen, and sulfur, q and r are, independently, integers from 0 to 4, provided that q and r cannot be 0 simultaneously, Z is an oligonucleoside moiety, Compound.

[0585] 88. A compound of formula (XVa).

[0586]

Chemical formula

[0587] 89. A compound of formula (XVb).

[0588]

Chemical formula

[0589] 90. Use of a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, for preparing a compound according to any one of propositions 76, 81 to 84, 87.

[0590] 91. Use of a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, wherein R2 = F, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, for preparing a compound according to proposition 85.

[0591] 92. Use of a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, wherein R2 = OH, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, for preparing a compound according to proposition 86.

[0592] 93. Use of a compound according to any of propositions 20, 25, 27, 29, 54, 56, and / or a composition according to any of propositions 30, 31, 57, 58, for preparing a compound according to proposition 77.

[0593] 94. Use of a compound according to any of propositions 20, 25, 28, 29, 55, 56, and / or a composition according to any of propositions 30, 31, 57, 58, for preparing a compound according to proposition 78.

[0594] 95. Use of a compound according to any of propositions 21, 26, 32, 34, 59, 61, and / or a composition according to any of propositions 35, 36, 62, 63, for preparing a compound according to proposition 79.

[0595] 96. Use of a compound according to any of propositions 21, 26, 33, 34, 60, 61, and / or a composition according to any of propositions 35, 36, 62, 63, for preparing a compound according to proposition 80.

[0596] 97. Use of a compound according to any of propositions 20, 25, 27 to 29, 54 to 56, and / or a composition according to any of propositions 30, 31, 57, 58, for preparing a compound according to proposition 88.

[0597] 98. Use of a compound according to any of propositions 21, 26, 32 to 34, 59 to 61, and / or a composition according to any of propositions 35, 36, 62, 63, for preparing a compound according to proposition 89.

[0598] 99. A compound or composition obtainable or obtainable by a method according to any of propositions 68 to 75.

[0599] A pharmaceutical composition comprising a compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0600] A compound according to any one of propositions 1 to 29, 32 to 34, 37 to 56, 59 to 61, and 64 to 67, and / or a composition according to any one of propositions 30, 31, 35, 36, 57, 58, 62, and 63 for use in therapy.

[0601] In another aspect, the present invention can be applied to the compounds, methods, compositions, or uses of item numbers 1 to 56 below (references to any formula in the items refer only to the formulas defined within items 1 to 56. Such formulas are reproduced in Figure 7).

[0602] 1. The following structure:

[0603]

Chemical formula

[0604] 2. The compound according to item 1, wherein s is an integer selected from 4 to 12.

[0605] 3. The compound according to item 2, wherein s is 6.

[0606] 4. The compound according to any one of items 1 to 3, wherein r is an integer selected from 4 to 14.

[0607] 5. The compound according to item 4, wherein r is 6.

[0608] 6. The compound according to item 4, wherein r is 12.

[0609] 7. The compound according to item 5, which is dependent on item 3.

[0610] 8. The compound according to item 6, which is dependent on item 3.

[0611] 9. Z is

[0612]

Chemical formula

[0613] 10. The compound according to any one of items 1 to 9, wherein the oligonucleoside is an RNA compound capable of modulating, preferably inhibiting, the expression of a target gene.

[0614] 11. The RNA compound includes an RNA duplex containing a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, and each of the first strand and the second strand has 5' and 3' ends. The compound according to item 10.

[0615] 12. The RNA compound is attached to an adjacent phosphate at the 5' end of its second strand, preferably, the compound according to item 11, which is also dependent on items 3 and 6.

[0616] 13. The RNA compound is the compound according to item 11, which is attached to the adjacent phosphate at the 3' end of its second strand, preferably also dependent on items 3 and 5.

[0617] 14. The compound of formula (II * ), preferably dependent on item 12.

[0618]

Chemical formula

[0619] 15. The compound of formula (III * ), preferably dependent on item 13.

[0620]

Chemical formula

[0621] 16. The oligonucleoside contains an RNA double strand further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position, and is the compound defined in any of items 1 to 15.

[0622] 17. The modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy, and is the compound according to item 16.

[0623] 18. The oligonucleoside further comprises one or more deprotecting moieties at one or more ends, and is the compound according to any of items 1 to 17.

[0624] 19. The one or more deprotection moieties are not present at the termini of the oligonucleoside chain carrying the linker / ligand moiety, and / or the one or more deprotection moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleosides are present in the distal strand of the same chain as the terminus carrying the linker / ligand moiety, the compound according to item 18.

[0625] 20. The ligand moiety illustrated in formula (I * ) of item 1 contains one or more ligands, the compound according to any one of items 1 to 19.

[0626] 21. The ligand moiety illustrated in formula (I * ) of item 1 contains one or more carbohydrate ligands, the compound according to item 20.

[0627] 22. The one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides, the compound according to item 21.

[0628] 23. The one or more carbohydrates contain one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties, the compound according to item 22.

[0629] 24. The one or more carbohydrates contain one or more N-acetyl-galactosamine moieties, the compound according to item 23.

[0630] 25. The compound according to item 24 containing two or three N-acetylgalactosamine moieties.

[0631] 26. The one or more ligands are attached in a linear or branched configuration, the compound according to any one of the preceding items.

[0632] 27. The compound according to item 26, wherein the one or more ligands are attached as a bifurcated or trifurcated branched chain structure.

[0633] 28. The moiety shown in formula (I * ) of item 1:

[0634]

Chemical formula

[0635]

Chemical formula

[0636]

Chemical formula

[0637]

Chemical formula

[0638] 29. The part shown in the formula (I * ) of item 1:

[0639]

Chemical formula

[0640]

Chemical formula

[0641] 30. The compound according to item 28 or 29, where a = 2.

[0642] 31. The compound according to item 28 or 29, where a = 3.

[0643] 32. The compound according to item 28, where b = 3.

[0644] 33. The compound of the formula (VIII * ).

[0645]

Chemical formula

[0646] 34. The compound of the formula (IX * ).

[0647]

Chemical formula

[0648] 35. The compound according to item 33 or 34, wherein the oligonucleoside comprises an RNA duplex further comprising one or more riboses modified at the 2'-position, preferably a plurality of riboses modified at the 2'-position.

[0649] 36. The compound according to item 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluoro, and 2'-deoxy.

[0650] 37. The compound according to any one of items 33 to 36, wherein the oligonucleoside further comprises one or more deprotecting moieties at one or more termini.

[0651] 38. The one or more deprotecting moieties are not present at the termini of the oligonucleoside chain carrying the linker / ligand moiety, and / or the one or more deprotecting moieties are selected from phosphorothioate internucleoside linkages, phosphorodithioate internucleoside linkages, and inverted abasic nucleosides, and the inverted abasic nucleoside is present at the distal end of the same chain as the terminus carrying the linker / ligand moiety. The compound according to item 37.

[0652] 39. The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' termini, and the RNA duplex is attached to an adjacent phosphate at the 5' terminus of its second strand. The compound according to item 33.

[0653] 40. The oligonucleoside comprises an RNA duplex comprising a first strand and a second strand, the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, each of the first strand and the second strand has 5' and 3' termini, and the RNA duplex is attached to an adjacent phosphate at the 3' terminus of its second strand. The compound according to item 34.

[0654] 41. A method for preparing a compound according to any one of items 1 to 40, the method comprising reacting a compound of formula (X * ) and (XI * ):

[0655]

Chemical formula

[0656] 42. The compound of formula (X * ) is of formula (Xa * ),

[0657]

Chemical formula

[0658]

Chemical formula

[0659] 43. The compound of formula (X * ) is of formula (Xb * ),

[0660]

Chemical formula

[0661]

Chemical formula

[0662] 44. The compound of formula (XIa * ) is of formula (XIb * ),

[0663]

Chemical formula

[0664] 45. Formula (X * ):

[0665]

Chemical formula

[0666] 46. A compound of formula (Xa * ).

[0667] [Chemical formula]

[0668] 47. A compound of formula (Xb * ).

[0669] [Chemical formula]

[0670] 48. A compound of formula (XI * ):

[0671] [Chemical formula] a compound of wherein s is independently an integer selected from 1 to 16, Z is an oligonucleoside moiety, compound.

[0672] 49. A compound of formula (XIa * ).

[0673] [Chemical formula]

[0674] 50. A compound of formula (XIb * ).

[0675]

Chem.

[0676] 51. Use of a compound according to any one of items 45 and 48 to 50 for preparing a compound according to any one of items 1 to 40.

[0677] 52. Use of a compound according to item 46 for preparing a compound according to any one of items 6, 8 to 14, 16 to 33, and 35 to 40.

[0678] 53. Use of a compound according to item 47 for preparing a compound according to any one of items 5, 7, 9 to 13, 15 to 32, and 34 to 40.

[0679] 54. A compound or composition obtainable or obtained by a method according to any one of items 41 to 44.

[0680] 55. A pharmaceutical composition comprising a compound according to any one of items 1 to 40 together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0681] A compound according to any one of items 1 to 40 for use in therapy. Examples

[0682] The present invention will be more fully understood by reference to the following examples. However, such examples should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art based on them, and it is understood that they are included within the spirit and scope of the present application and within the scope of the appended claims.

Examples

[0683] Target identification Background All biological functions, including hemostasis (physiological control of blood loss), result from the coordinated interactions of hundreds of interacting molecules, mainly proteins, and can be considered emergent functional consequences of protein - protein interaction networks, where each node of the network is a protein and each edge connecting proteins can represent a series of possible interaction types ranging from complex formation to catalytic activation.

[0684] Historically, such functions have been represented as simplified linear pathways. However, as knowledge has increased, it has become clear that the level of complexity that can adequately represent the functional properties of biological processes, capture the resilience to perturbations, and the robustness to random damage of individual components is a network, as pathways are more complex.

[0685] Networks that underlie biological functions may consist of several interacting canonical pathways, as well as additional proteins that are mainly involved when canonical functions are disrupted. Therefore, it is important to develop methods that can model this complexity in a meaningful and tractable way, and to generate target hypotheses that take into account the inherent resistance to change, which is a consequence of network robustness. An example of the additional complexity captured by this framework is shown in Figure 8.

[0686] The over - simplification of biological features and the failure to select rational drug targets based on a realistic model of biological processes are contributing factors to the poor success rate of drug discovery. Furthermore, there is a lack of rigorous and objective methods for creating network models of processes and for prioritizing protein targets within such models. As a result, target - selection decisions are often made ad - hoc based on priorities or evidence unrelated to functional models.

[0687] Hemostasis is a complex process involving multiple cell types and proteins, and is tightly regulated by systems of pro-hemostatic and anti-hemostatic mechanisms. Hemophilia and other hemorrhagic disorders involve genetic defects in individual protein components that affect the hemostatic system's ability to control blood loss to varying degrees. There is a large unmet need for therapies that improve hemostasis and are patient-friendly, well-tolerated, and safe.

[0688] Methods for identifying processes and targets The inventors used network biology approaches to create a network model of hemostasis (Figure 9). The network model is designed to capture all the important proteins involved in the process, as well as their connectivity and, importantly, the direction of information flow between protein pairs.

[0689] The inventors analyzed such network models using their own analysis methods. In such methods, direction information is used to capture important "target" characteristics such as whether a protein is an information integration factor, whether it is an important pathway for information to other parts of the network, whether it is an influencing factor for important proteins, and the degree to which an influencing factor is affected or affects other proteins (based on the absolute and relative numbers and directions of inputs and outputs). Direction information also makes it possible to infer the hierarchical relationships between proteins. Proteins higher in the hierarchy and having a particular characteristic may be more preferable than proteins having similar characteristics otherwise. By comparing the relative specificity and magnitude of each characteristic with other characteristics, the inventors were able to score and rank proteins in terms of their target suitability.

[0690] The ability to characterize such target characteristics from the perspective of network relatedness makes it possible to judge the selectivity and magnitude of the effects in a selected situation and thus the suitability of each for a given indication.

[0691] As a result, the inventors were able to identify targets that would provide improved blood loss control in hemophilia and other hemorrhagic disorders. The analysis used for this purpose was specially tailored to find a novel and non-obvious indirect enhancer of blood loss, in which knockdown by GalNAc-siRNA in hepatocytes suppresses the production of secreted proteins that play a regulatory role in hemostasis and thus would be beneficial in hemophilia.

[0692] Specifically, a set of directed network models was algorithmically generated using a selected set of hemostasis-related pathway proteins and an internal proprietary database of protein-protein relationships that includes the "directionality" of the interactions. This direction information was derived from a series of public and internal databases complemented by directionality estimated from natural language processing of the text of scientific publications. The inventors' network construction algorithm used these information sources to build "directed" models of important biological processes.

[0693] Subsequently, proprietary analysis techniques were applied to the network models to identify pharmacologically promising targets within the network. Their knockdown would have a significant impact on the network and thus on the modeled biological functions. Such algorithms extensively utilize direction information and hierarchical relationships to identify targets with a set of specific properties that would thereby make them good siRNA targets. The targets were then further filtered by protein class and hepatocyte specificity according to therapeutic needs.

[0694] The above workflow leveraging proprietary data resources and network node metrics has identified two clinically validated hemostasis modulators (AT3 shown above being one of them) under development for treating hemorrhagic shock. Additionally, the above workflow highlights a novel and non-obvious coagulation modulator, Protein Z-dependent protease inhibitor (ZPI), which is predicted by our analysis to be superior to any based on metrics that estimate indirect effects and thus potentially have an excellent safety profile (Figure 9).

Example

[0695] Synthesis of Tether 1 Basic experimental conditions: Thin layer chromatography (TLC) was performed on silica-coated aluminum plates using Macherey-Nagel's 254 nm fluorescent indicator. Compounds were visualized under UV light (254 nm) or after spraying with 5% H2SO4 or ninhydrin reagent in methanol (MeOH) according to Stahl (from Sigma-Aldrich) followed by heating. Flash chromatography was performed on a Biotage Isolera One flash chromatography instrument equipped with a dual variable UV wavelength detector (200 - 400 nm) using Biotage Sfar silica 10, 25, 50, or 100 g columns (Uppsala, Sweden).

[0696] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich and solvents from Carl Roth GmbH+Co.KG. D-Galactosamine pentaacetate was purchased from AK scientific.

[0697] HPLC / ESI-MS was performed using a Waters Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1×100 mm) at 60 °C on a Dionex UltiMate 3000RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A, which was H2O containing 0.1% formic acid, and solvent B, which was acetonitrile (ACN) containing 0.1% formic acid. A gradient from 5% to 100% B was used at a flow rate of 0.4 mL / min for 15 min. Detector and conditions: Corona ultra charged aerosol detection (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0698] 1 H and 13 C NMR spectra were recorded at room temperature on a Varian spectrometer operating at 500 MHz ( 1 H NMR) and 125 MHz ( 13 C NMR). Chemical shifts are reported in ppm relative to the solvent residual peaks (CDCl3 - 1 H NMR: δ 7.26 ppm and 13 C NMR δ 77.2 ppm; DMSO-d6 - 1 H NMR: δ 2.50 ppm and 13 C NMR δ 39.5 ppm). Coupling constants are reported in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[0699] Synthetic route for the conjugate building block TriGalNAc_tether1:

[0700]

Chem.

[0701] Preparation of Compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 equiv) was added. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aqueous NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the title compound as a yellow oil. It was purified by flash chromatography (gradient elution: 0 - 10% MeOH in DCM, 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[0702] [Chemical Structure]

[0703] Preparation of Compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 equiv) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 equiv) were dissolved in anhydrous DCM (40 mL) under argon, and molecular sieves 3 Å (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. Then TMSOTf (0.77 g, 3.49 mmol, 0.5 equiv) was added to the mixture, and the reaction was stirred overnight. The molecular sieves were filtered off, the filtrate was diluted with DCM (100 mL), and washed with cold saturated aqueous NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0 - 3% MeOH in DCM, 10 CV) to give the title product as a bright yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: C 20 H 32 N4O 11 Calculated for, 504.21. Found 505.4. 11H 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 13C NMR (125 MHz, CDCl3) δ 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).

[0704]

Chem.

[0705] Preparation of Compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 equiv) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1:1 volume / volume), and Pd / C (100 mg) was added. The reaction mixture was degassed by vacuum / argon cycle (3 times) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to obtain the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). This compound was used without further purification. MS: C 20 H 34 N2O 11 calculated value, 478.2. Found 479.4.

[0706]

Chem.

[0707] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 equiv) was dissolved in a mixture of DCM / water (40 mL 1:1 volume / volume), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 equiv) was added with vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 equiv) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure, and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% ethyl acetate in cyclohexane, 12 CV) to give the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: C 33 H 53 NO 11 Calculated for, 639.3. Found 640.9. 1 H NMR (500 MHz, DMSO-d6) δ 7.38 - 7.26 (m, 5H), 4.97 (s, 2H), 3.54 (t, 6H), 3.50 (s, 6H), 2.38 (t, 6H), 1.39 (s, 27H). 13 C NMR (125 MHz, DMSO-d6) δ 170.3 (3xC), 154.5 (C), 137.1 (C), 128.2 (2xCH), 127.7 (CH), 127.6 (2xCH), 79.7 (3xC), 68.4 (3xCH2), 66.8 (3xCH2), 64.9 (C), 58.7 (CH2), 35.8 (3xCH2), 27.7 (9xCH3).

[0708]

Chemical Structure

[0709] Preparation of Compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 equiv) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated three times with toluene (5 mL) and dried under high vacuum to give the compound as the TFA salt (0.183 g, 98%). This compound was used without further purification. MS: C 21 H 29 NO 11 Calculated for, 471.6. Found 472.4.

[0710]

Chem.

[0711] Preparation of Compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 equiv) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 equiv) were dissolved in N,N-dimethylformamide (DMF) (25 mL). Then, N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 equiv), 1-hydroxybenzotriazole hydrate (HoBt) (1.05 g, 7.44 mmol, 5.0 equiv) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 equiv) were added to this solution, and the reaction mixture was stirred for 72 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL), and washed with saturated aqueous NaHCO3 (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, 14 CV). The product was obtained as a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: C 81 H 125 N7O 41 Calculated for, 1852.9. Found 1854.7. 11H 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 13C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[0712]

Chem.

[0713] Preparation of Compound 10: The trifurcated GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 equiv) was dissolved in MeOH (15 mL), and 3 drops of acetic acid (AcOH) and Pd / C (30 mg) were added. The reaction mixture was degassed by vacuum / argon cycle (3 times) and hydrogenated overnight under balloon pressure. Mass spectrometry was performed following the completion of the reaction, and the resulting mixture was filtered through a thin pad of celite. The solvent was evaporated, and the obtained residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellow oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 Calculated value for, 1718.8. Observed value 1719.3.

[0714]

Chem.

[0715] Preparation of Compound 11: The commercially available bis(N-hydroxysuccinimide ester) of suberic acid (3.67 g, 9.9 mmol, 1.0 equiv) was dissolved in DMF (5 mL), and triethylamine (1.2 mL) was added. To this solution, a solution of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0 equiv) in DMF (5 mL) was added dropwise. The reaction was stirred at room temperature for 3 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 16 CV). The product was obtained as a white solid (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM)). MS: C 15 H 23 Calculated value for N5O5, 353.4. Observed value 354.3.

[0716]

Chem.

[0717] Preparation of TriGalNAc(12): The branched GalNAc compound 10 (0.35 g, 0.24 mmol, 1.0 equivalent) and compound 11 (0.11 g, 0.31 mmol, 1.5 equivalents) were dissolved in DCM (5 mL) under argon, and triethylamine (0.1 mL, 0.61 mmol, 3.0 equivalents) was added. The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was evaporated, and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% MeOH in DCM, 20 CV) to obtain the title compound as a white, fluffy solid (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)). MS: C 84 H 137 N 11 O 41 Calculated value for, 1957.1. Found 1959.6.

[0718] Conjugation of the tether 1 with the siRNA strand: Monofluoro cyclooctyne (MFCO) conjugation at the 5'- or 3'-terminus 5'-terminal MFCO conjugation

[0719]

Chem.

[0720]

Chem.

[0721] Basic conditions for MFCO conjugation: The amine-modified single strand was dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer pH 9.6 / dimethyl sulfoxide (DMSO) 4:6 (volume / volume), and to this solution was added 1 molar equivalent of a DMF solution of 35 mM MFCO-C6-NHS ester (Berry & Associates, catalog number LK4300). The reaction was carried out at room temperature, and after 1 hour another 1 molar equivalent of the MFCO solution was added. The reaction was allowed to proceed for a further 1 hour and monitored by LC / MS. To achieve quantitative consumption of the starting material, at least 2 molar equivalents in excess of the MFCO NHS ester reagent were required relative to the amino-modified oligonucleotide. The reaction mixture was diluted 15-fold with water, filtered through a Sartorius 1.2 μm filter, and then purified by reverse phase (RP HPLC) using an Akta Pure instrument (GE Healthcare).

[0722] Purification was carried out using a Waters XBridge C18 Prep 19×50 mm column. Buffer A was 100 mM TEAAc pH 7, and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60 °C were used. A UV trace at 280 nm was recorded. A gradient of 0 - 100% B was used within 60 column volumes.

[0723] Fractions containing the full-length conjugate oligonucleotide were pooled, precipitated with 3M NaOAc, pH 5.2 and 85% ethanol in a freezer, and the collected pellet was dissolved in water. The sample was desalted by size exclusion chromatography, concentrated using a speed-vac concentrator, and the conjugate oligonucleotide was obtained in an isolated yield of 40 - 80%. 5’-GalNAc-T1 conjugate

[0724]

Chemical formula

[0725] [Chemistry]

[0726] Basic procedure of TriGalNAc conjugation: The MFCO-modified single strand was dissolved in water at 2000 OD / mL, and 1 equivalent of the DMF solution of Compound 12 (10 mM) was added to this solution. The reaction was carried out at room temperature, and after 3 hours, 0.7 molar equivalent of the Compound 12 solution was added. The reaction was allowed to proceed overnight and monitored for completion by LCMS. The conjugate was diluted 15-fold with water, filtered through a Sartorius 1.2 μm filter, and then purified by RP-HPLC on an Akta Pure instrument (GE Healthcare).

[0727] RP-HPLC purification was carried out using a Waters XBridge C18 Prep 19×50 mm column. Buffer A was 100 mM triethylammonium acetate pH 7, and Buffer B contained 95% acetonitrile in Buffer A. A flow rate of 10 mL / min and a temperature of 60 °C were used. A UV trace at 280 nm was recorded. A gradient of 0-100% B was used within 60 column volumes.

[0728] Fractions containing the full-length conjugate oligonucleotide were pooled, precipitated with 3M NaOAc, pH 5.2 and 85% ethanol in a freezer, and the collected pellet was dissolved in water to obtain an oligonucleotide solution of approximately 1000 OD / mL. O-acetate was removed by adding 20% aqueous ammonia. Quantitative removal of such protecting groups was verified by LC-MS.

[0729] The conjugate was desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to obtain the conjugate oligonucleotide in a 50-70% isolated yield.

[0730] The following scheme further shows the synthetic route.

[0731]

Chem.

[0732]

Chem.

[0733]

Chem.

[0734]

Chem.

[0735]

Chem.

Example

[0736] Double-strand annealing To generate the desired siRNA double strand, two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixture was placed in a water bath at 70 °C for 5 minutes, and then allowed to cool to ambient temperature within 2 hours. The double strand was lyophilized for 2 days and stored at -20 °C.

[0737] The double-stranded chains were analyzed by analytical SEC HPLC using a Superdex™ 75 Increase 5 / 150 GL column 5 × 153 - 158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC instrument. The mobile phase consisted of 1×PBS containing 10% acetonitrile. An isocratic gradient was run at a flow rate of 1.5 mL / min for 10 minutes at room temperature. UV traces at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma-Aldrich and phosphate-buffered saline (PBS; 10×, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

Example

[0738] Synthesis of Tether 2 Basic experimental conditions: Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using Macherey-Nagel's 254 nm fluorescent indicator. Compounds were visualized under UV light (254 nm) or after spraying with 5% H2SO4 or ninhydrin reagent in methanol (MeOH) according to Stahl (from Sigma-Aldrich) followed by heating. Flash chromatography was performed using Biotage Sfar silica 10, 25, 50, or 100 g columns (Uppsala, Sweden) on a Biotage Isolera One flash chromatography instrument equipped with a dual variable UV wavelength detector (200 - 400 nm).

[0739] All humidity-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich and the solvents were purchased from Carl Roth GmbH+Co.KG. D-Galactosamine pentaacetate was purchased from AK scientific.

[0740] HPLC / ESI-MS was performed using a Waters Acquity UPLC Protein BEH C4 column (300 Å, 1.7 μm, 2.1 × 100 mm) at 60 °C on a Dionex UltiMate 3000RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A, which was H2O containing 0.1% formic acid, and solvent B, which was acetonitrile (ACN) containing 0.1% formic acid. A gradient of 5% to 100% B was used over 15 minutes at a flow rate of 0.4 mL / min. Detector and conditions: Corona ultra charged aerosol detection (from esa). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0741] 1 H and 13 C NMR spectra were recorded at room temperature on a Varian spectrometer operating at 500 MHz ( 1 H NMR) and 125 MHz ( 13 C NMR). Chemical shifts are reported in ppm relative to the solvent residual peak (CDCl3 - 1 H NMR: δ 7.26 ppm and 13 C NMR δ 77.2 ppm; DMSO-d6 - 1 H NMR: δ 2.50 ppm and 13 C NMR δ 39.5 ppm). Coupling constants are reported in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[0742] Synthetic route of conjugate building block TriGalNAc_tether2:

[0743]

Chem.

[0744] Preparation of Compound 2: D-Galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 equiv) was added. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aqueous NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the title compound as a yellow oil. It was purified by flash chromatography (gradient elution: 0 - 10% MeOH in DCM, 10 CV). The product was obtained as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)).

[0745]

Chem.

[0746] Preparation of Compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 equiv) and azido-PEG3-OH (1.83 g, 10.5 mmol, 1.5 equiv) were dissolved in anhydrous DCM (40 mL) under argon, and molecular sieves 3 Å (5 g) were added to the solution. The mixture was stirred at room temperature for 1 h. Then TMSOTf (0.77 g, 3.49 mmol, 0.5 equiv) was added to the mixture, and the reaction was stirred overnight. The molecular sieves were filtered off, the filtrate was diluted with DCM (100 mL), and washed with cold saturated aqueous NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude material was purified by flash chromatography (gradient elution: 0 - 3% MeOH in DCM, 10 CV) to give the title product as a bright yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)). MS: C 20 H 32 N4O 11 Calculated for, 504.21. Found 505.4. 11H 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 13C NMR (125 MHz, CDCl3) δ 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).

[0747]

Chem.

[0748] Preparation of Compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 equiv) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL, 1:1 volume / volume), and Pd / C (100 mg) was added. The reaction mixture was degassed by vacuum / argon cycle (3 times) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through celite and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to obtain the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). This compound was used without further purification. MS: C 20 H 34 N2O 11 calculated value, 478.2. Found 479.4.

[0749]

Chem.

[0750] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 equiv) was dissolved in a mixture of DCM / water (40 mL 1:1 volume / volume), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 equiv) was added with vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 equiv) was added dropwise to the mixture, and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was removed under reduced pressure, and the resulting crude material was purified by flash chromatography (gradient elution: 0 - 10% ethyl acetate in cyclohexane, 12 CV) to give the title compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)). MS: C 33 H 53 NO 11 Calculated for, 639.3. Found 640.9. 1 H NMR (500 MHz, DMSO-d6) δ 7.38 - 7.26 (m, 5H), 4.97 (s, 2H), 3.54 (t, 6H), 3.50 (s, 6H), 2.38 (t, 6H), 1.39 (s, 27H). 13 C NMR (125 MHz, DMSO-d6) δ 170.3 (3xC), 154.5 (C), 137.1 (C), 128.2 (2xCH), 127.7 (CH), 127.6 (2xCH), 79.7 (3xC), 68.4 (3xCH2), 66.8 (3xCH2), 64.9 (C), 58.7 (CH2), 35.8 (3xCH2), 27.7 (9xCH3).

[0751]

Chemical Structure

[0752] Preparation of Compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 equiv) was dissolved in CH2Cl2 (1 mL) under argon, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene (5 mL) three times and dried under high vacuum to obtain the compound as the TFA salt (0.183 g, 98%). This compound was used without further purification. MS: C 21 H 29 NO 11 Calculated value for, 471.6. Found 472.4.

[0753]

Chemical Structure

[0754] Preparation of Compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 equiv) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 equiv) were dissolved in N,N-dimethylformamide (DMF) (25 mL). Then, N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 equiv), 1-hydroxybenzotriazole hydrate (HoBt) (1.05 g, 7.44 mmol, 5.0 equiv) and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 equiv) were added to this solution, and the reaction was stirred for 72 h. The solvent was removed under reduced pressure, the residue was dissolved in DCM (100 mL), and washed with saturated aqueous NaHCO3 solution (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude product was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 14 CV). The product was obtained as a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: C 81 H 125 N7O 41 Calculated value for, 1852.9. Found 1854.7. 11H 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 13C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7 (CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[0755]

Chem.

[0756] Preparation of Compound 10: The trifurcated GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 equiv) was dissolved in MeOH (15 mL), and 3 drops of acetic acid (AcOH) and Pd / C (30 mg) were added. The reaction mixture was degassed by vacuum / argon cycle (3 times) and hydrogenated overnight under balloon pressure. Mass spectrometry was performed following the completion of the reaction, and the resulting mixture was filtered through a thin pad of celite. The solvent was evaporated, and the obtained residue was dried under high vacuum and used in the next step without further purification. The product was obtained as a pale yellow oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 Calculated value for, 1718.8. Measured value 1719.3.

[0757]

Chemical Structure

[0758] Preparation of Compound 14: The trifurcated GalNAc compound 10 (0.45 g, 0.26 mmol, 1.0 equiv), HBTU (0.19 g, 0.53 mmol, 2.0 equiv) and DIPEA (0.23 mL, 1.3 mmol, 5.0 equiv) were dissolved in DCM (10 mL) under argon. To this mixture, a solution of compound 13 (0.14 g, 0.53 mmol, 2.0 equiv) in DCM (5 mL) was added dropwise. The reaction was stirred at room temperature overnight. The solvent was removed, and the residue was dissolved in EtOAc (50 mL), washed with water (50 mL) and dried over Na2SO4. The solvent was evaporated, and the crude material was purified by flash chromatography (gradient elution: 0 - 5% MeOH in DCM, 20 CV). The product was obtained as a white, fluffy solid (0.25 g, 48%, rf = 0.4 (10% MeOH in DCM)). MS: C 88 H 137 N7O 42 Calculated value for, 1965.1. Measured value 1965.6.

[0759]

Chemical Structure

[0760] Preparation of TriGalNAc(15): The branched GalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 equivalent) was dissolved in EtOAc (15 mL), and Pd / C (40 mg) was added. The reaction mixture was degassed by vacuum / argon cycle (3 times) and hydrogenated overnight under balloon pressure. The completion of the reaction was monitored by mass spectrometry, and the resulting mixture was filtered through a thin pad of celite. The solvent was removed under reduced pressure, and the obtained residue was dried under high vacuum overnight. The residue was used for conjugation with oligonucleosides without further purification (0.28 g, quantitative yield). MS: C 81 H 131 N7O 42 Calculated value of, 1874.9. Measured value 1875.3.

[0761] Conjugation of tether 2 and siRNA strand: TriGalNAc tether 2 (GalNAc-T2) conjugation at the 5'-end or 3'-end 5'-GalNAc-T2 conjugate

[0762]

Chem.

[0763]

Chem.

[0764] Preparation of TriGalNAc tether 2 NHS ester: To a solution of carboxylic acid tether 2 (Compound 15, 227 mg, 121 μmol) in DMF (2.1 mL), N-hydroxysuccinimide (NHS) (15.3 mg, 133 μmol) and N,N'-diisopropylcarbodiimide (DIC) (19.7 μL, 127 μmol) were added. The solution was stirred at room temperature for 18 hours and used for the subsequent conjugation reaction without purification.

[0765] Basic conditions for triGalNAc tether 2 conjugation: The amine-modified single strand was dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer pH 9.6 / DMSO 4:6 (volume / volume). To this solution, a 1 molar equivalent solution of tether 2 NHS ester (57 mM) in DMF was added. The reaction was carried out at room temperature and after 1 hour another 1 molar equivalent of the NHS ester solution was added. The reaction was allowed to proceed for a further 1 hour and the progress of the reaction was monitored by LCMS. To achieve quantitative consumption of the starting material, at least a 2 molar equivalent excess of the NHS ester reagent was required relative to the amino-modified oligonucleotide. The reaction mixture was diluted 15-fold with water, filtered through a Sartorius 1.2 μm filter and then purified by reverse phase (RP HPLC) on an Akta Pure (GE Healthcare) instrument.

[0766] Purification was carried out using a Waters XBridge C18 Prep 19×50 mm column. Buffer A was 100 mM TEAA pH 7 and buffer B contained 95% acetonitrile in buffer A. A flow rate of 10 mL / min and a temperature of 60 °C were used. A UV trace at 280 nm was recorded. A gradient of 0 - 100% B was used within 60 column volumes.

[0767] Fractions containing the full-length conjugate oligonucleotide were pooled and precipitated with 3M NaOAc, pH 5.2 and 85% ethanol in a freezer and then dissolved in water at 1000 OD / mL. The O-acetate was removed with 20% aqueous ammonium hydroxide until completion (monitored by LC-MS).

[0768] The conjugate was desalted by size exclusion chromatography using Sephadex G25 Fine resin (GE Healthcare) on an Akta Pure (GE Healthcare) instrument to obtain the conjugate oligonucleotide in 60 - 80% isolated yield.

[0769] The conjugate was characterized by HPLC-MS analysis using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics) and a 2.1×50 mm XBridge C18 column (Waters). Buffer A was 16.3 mM triethylamine, 100 mM HFIP in 1% aqueous MeOH, and buffer B contained 95% MeOH in buffer A. A flow rate of 250 μL / min and a temperature of 60 °C were used. UV traces at 260 and 280 nm were recorded. A gradient of 1-100% B was used within 31 minutes.

[0770] The following scheme further shows the synthetic route.

[0771]

Chem.

[0772]

Chem.

[0773]

Chem.

[0774]

Chem.

Example

[0775] Double-strand annealing To generate the desired siRNA double strand, two complementary strands were annealed by combining equimolar aqueous solutions of both strands. The mixture was placed in a 70 °C water bath for 5 minutes, followed by allowing cooling to ambient temperature within 2 hours. The double strand was lyophilized for 2 days and stored at -20 °C.

[0776] The double-strand was analyzed by analytical SEC HPLC using a Superdex™ 75 Increase 5 / 150 GL column 5 × 153 - 158 mm (Cytiva) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC instrument. The mobile phase consisted of 1×PBS containing 10% acetonitrile. An isocratic gradient was run at a flow rate of 1.5 mL / min for 10 minutes at room temperature. UV traces at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS; 10×, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

Example

[0777] Alternative synthetic route for conjugate building block TriGalNAc_tether2:

[0778]

Chemical formula

[0779] Conjugation of tether2 with siRNA strand: TriGalNAc tether2 (GalNAc-T2) conjugation at the 5'-end or 3'-end Conjugation conditions

[0780]

Chemical formula

[0781]

Chemical Structure

[0782]

Chemical Structure

Example

[0783] Solid-phase synthesis method: scale ≤ 1 μMOL The synthesis of the siRNA sense and antisense strands was carried out using a commercially available solid support made of porous controlled glass with a universal linker (universal CPG, loading 40 μmol / g; LGC Biosearch or Glen Research) on a MerMade192X synthesizer.

[0784] RNA phosphoramidites were purchased from ChemGenes or Hongene.

[0785] The 2'-O-methyl phosphoramidites used were as follows: 5'-(4,4'-dimethoxytrityl)-N-benzoyl-adenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-cytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0786] The 2'-F-phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxythymidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0787] All phosphoramidites, except for 2'-O-methyl-uridine phosphoramidite dissolved in DMF / MeCN (1:4, volume / volume), were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M. Iodine (DNAchem) at 0.02 M in acetonitrile / pyridine / H2O was used as the oxidizing reagent. Thiolation of phosphorothioate linkages was carried out using PADS (TCI) at 0.2 M in acetonitrile / pyridine 1:1 volume / volume. 5-Ethylthiotetrazole (ETT) at 0.25 M mM in acetonitrile was used as the activator solution.

[0788] The inverted deoxy base phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[0789] In each cycle, DMT was removed with a deblocking solution, 3% TCA in DCM (DNAchem).

[0790] The coupling time was 180 seconds. The oxidant contact time was set to 80 seconds, and the thiolation time was 2 * 100 seconds.

[0791] At the end of the synthesis, an NH4OH:EtOH solution 4:1 (volume / volume) (TCI) was used at 45 °C for 20 hours to cleave the oligonucleotide from the solid support. The solid support was then filtered, the filter was washed well with H2O, and the volume of the mixed solution was reduced by evaporation under reduced pressure.

[0792] The oligonucleotide was processed using an Amicon Ultra-2 centrifugal filter unit; by ultracentrifugation using PBS buffer (10×, Teknova, pH 7.4, sterile), or by EtOH precipitation from 1 M sodium acetate, to form the sodium salt.

[0793] The identity of the single strand was evaluated by MS ESI, then annealed in water to form the final double-stranded siRNA, and the double-stranded purity was evaluated by size exclusion chromatography.

Example

[0794] Solid-phase synthesis method: Scale ≥ 5 μMOL The synthesis of the siRNA sense strand and antisense strand was carried out on a commercially available solid support made of porous controlled glass with a universal linker (universal CPG, loading 40 μmol / g; LGC Biosearch or Glen Research) at a scale of 5 μmol using a MerMade12 synthesizer. The sense strand for the purpose of 3'-conjugation was synthesized at 12 μmol on a 3'-PT-amino modified substance C6 CPG 500 Å solid support (LGC) with a loading of 86 μmol / g.

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

[0796] The 2'-O-methyl phosphoramidites used were as follows: 5'-(4,4'-dimethoxytrityl)-N-benzoyl-adenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-cytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-guanosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-uridine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0797] The 2'-F-phosphoramidites used were as follows: 5'-dimethoxytrityl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-dimethoxytrityl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-dimethoxytrityl-deoxythymidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0798] The inverted abasic phosphoramidite, 3-O-dimethoxytrityl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, was purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[0799] All phosphoramidites except 2'-O-methyl-uridine phosphoramidite dissolved in DMF / MeCN (1:4, volume / volume) were dissolved in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M. Iodine (DNAchem) at 0.02 M in acetonitrile / pyridine / H2O was used as the oxidizing reagent. Thiolation of phosphorothioate linkages was carried out using 0.2 M PADS (TCI) in acetonitrile / pyridine 1:1 volume / volume. 5-Ethylthiotetrazole (ETT) at 0.25 M mM in acetonitrile was used as the activator solution.

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

[0801] In the case of the chain synthesized with universal CPG, coupling was carried out for 130 seconds using 8 equivalents of amidite. The oxidation time was 47 seconds and the thiolation time was 210 seconds.

[0802] In the case of the chain synthesized with 3'-PT-amino-modified substance C6 CPG, coupling was carried out for 2 * 150 seconds using 8 equivalents of amidite. The oxidation time was 47 seconds and the thiolation time was 250 seconds.

[0803] At the end of the synthesis, the oligonucleotide was cleaved from the solid support using a 4:1 (volume / volume) NH4OH:EtOH solution (TCI) at 45 °C for 20 hours. The solid support was then filtered, the filter was washed well with H2O, and the volume of the mixed solution was reduced by evaporation under reduced pressure.

[0804] The oligonucleotide was treated by ethanol precipitation from 1M sodium acetate to form the sodium salt.

[0805] The single-stranded oligonucleotide was purified by IP-RP HPLC on an Xbridge BEH C18 5μm, 130Å, 19×150mm (Waters) column using a gradient of B in A. Mobile phase A: 240 mM HFIP, 7 mM TEA, and 5% methanol in water; mobile phase B: 240 mM HFIP, 7 mM TEA in methanol.

[0806] The single-stranded purity and identity were evaluated by UPLC / MS ESI- on an Xbridge BEH C18 2.5μm, 3×50mm (Waters) column using a 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 (volume / volume).

[0807] The sense strand was conjugated according to the protocol provided in any of Examples 2, 4, 6.

[0808] Next, the sense strand and the antisense strand were annealed in water to form the final double-stranded siRNA, and the double-strand purity was evaluated by size exclusion chromatography.

[0809] The present invention is not intended to be limited in scope to the specific disclosed embodiments provided, for example, to illustrate various aspects of the present invention. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such variations can be made without departing from the true scope and spirit of the present disclosure and are intended to fall within the scope of the present disclosure.

Example

[0810] In vivo efficacy data in a hemophilia mouse model In this study, 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 wild-type (WT) mice were used.

[0811]

Table 9

[0812] Eight days before inducing knee blood loss, the GalNAc-siRNA construct ETXM1184 or vehicle (0.9% saline) was injected subcutaneously (s.c.) into the mice at a dose volume of 5 ml / kg.

[0813] To induce knee blood loss, the weight of the mice was measured and they were anesthetized using isoflurane inhalation anesthetic. Both legs were shaved and the knee joints were exposed. For analgesia, 10 ml / kg of buprenorphine was injected s.c. into the mice, and the diameters of both knees were measured with an electronic caliper. Subsequently, both knees were wiped with 70% ethanol.

[0814] A 30G sterile subcutaneous needle was inserted into the infrapatellar ligament of one knee. The knee to be injected was randomized left or right, and the injected side was recorded. The mice were removed from anesthesia and allowed to recover in a warming cage before being returned to their original cage.

[0815] The mice were monitored regularly over the first 6 hours, and 10 ml / kg of buprenorphine was injected subcutaneously for analgesia 6 hours after injury. The visual blood loss score (VBS) of the injured knee was evaluated 72 hours and 10 days after injury.

[0816] All mice were carefully examined daily for clinical signs of excessive blood loss. Mice showing clinical signs of excessive blood loss, piloerection, withdrawal from cage mates, or facial grimacing were euthanized for welfare reasons.

[0817] The mice were removed from the study 10 days after injury.

[0818] Citrate - added blood samples were collected by cardiac puncture under isoflurane anesthesia, plasma was prepared, and aliquots were frozen in dry ice and stored at - 80°C. For this purpose, blood was collected and placed in 3.8% sodium citrate in a 1:9 ratio, followed by centrifugation at 7000×g for 10 minutes at 4°C. Specifically, the following steps were performed. 1. Collect blood by cardiac puncture. 2. Flush the syringe and needle with sodium citrate solution (3.8%) and leave the solution in the hub of the syringe (about 30 μl). 3. After blood collection, the sample is discharged into a 1.5 ml microcentrifuge tube, and sufficient sodium citrate solution (3.8%) is added to ensure an exact sodium citrate:blood ratio of 1:9. The sodium citrate solution is added not directly to the sample but to the side of the tube. Invert 4 - 6 times to mix. If the sample is not centrifuged immediately, store it in a refrigerator if available, or alternatively on a wrapped ice block, and continue to invert the collection tube regularly. 4. Centrifuge the sample as soon as possible at 4°C for 10 minutes at a rotational speed of 7000×g. 5. Remove all the plasma from the sample and transfer it to a new microcentrifuge tube. 6. Dispense the plasma into pre - labeled tubes (Thermo Scientific, 10775974) as follows. · 30 μl for potential TGA assay · 100 μl for potential APTT assay · The rest is all for potential target protein abundance analysis. 7. Place all the aliquots immediately on wet ice / dry ice. 8. Transport the sample while it is placed on wet ice / dry ice. 9. Transfer the sample to a - 20°C / -80°C freezer and store it until use.

[0819] The liver was removed, and at most three parts of each lobe were placed in RNA later and stored at 4°C for 24 - 72 hours. Then the tissue was blotted dry, weighed, and stored at - 80°C. Specifically, the following steps were carried out. 1. Immediately after cardiac puncture, euthanize the mouse by cervical dislocation. 2. Incise the abdominal wall and remove the liver as quickly as possible. 3. Place the liver on a Petri dish on wet ice to minimize sample degradation. 4. From each of the following lobes: left lobe, middle lobe, right lobe, and caudate lobe, cut 3 × approximately 50 mg liver slices. Immediately place these liver slices into pre-labeled tubes (1.5 ml microcentrifuge tubes) containing 500 μl of RNAlater, and place the collection tubes on wet ice. a. Transport while on wet ice and transfer to 4 °C for storage. b. After 24 - 72 hours, blot the liver samples and measure the weight. Record the weight on a terminal sheet. c. Transfer to -80 °C for long-term storage. 5. Collect any remaining liver and place it into another pre-labeled collection tube (2 ml microcentrifuge tube). a. Freeze with dry ice for potential future analysis. b. Transport the samples while on dry ice. c. Transfer to -80 °C for long-term storage. 6. Clean all dissection instruments between animals to prevent any cross-contamination.

[0820] The skin was removed from the leg and the knee joint was measured. Subsequently, the leg was immersed in 10% formalin before decalcification and slide preparation. Specifically, the following steps were performed. 1. After removing the liver, measure and record the diameters of both the injured and uninjured knees. 2. Remove the skin from both knees. Perform a visual blood loss score and measure the knee joint. 3. Dissect the leg from the upper femur to the ankle joint, removing some of the excess muscle while taking care not to cause any damage to the knee and associated structures. Place the knee into a pre-labeled tube (7 ml bijou tube) containing 10% neutral buffered formalin for processing for histological analysis.

[0821] On both the 3rd and 10th days after knee blood loss induction, hemA mice that received the GalNAc-siRNA construct ETXM1184 showed a significant reduction in the visual blood loss score compared to hemA mice that received the vehicle (0.9% saline) (see Figures 10A and 10B). Furthermore, the knee diameter of mice that received the GalNAc-siRNA construct ETXM1184 recovered more rapidly after knee blood loss induction compared to mice that received the vehicle (Figure 11A). This observation was confirmed by comparing the difference between the injured skin detachment knee diameter and the uninjured skin detachment knee diameter (Figure 11B).

[0822] Analysis of hemA mice 10 days after injury revealed that in mice that received the GalNAc-siRNA construct ETXM1184, the severity of myeloid hyperplasia was lower (Figure 12A), the severity of osteoarthritis was lower (Figure 12B), the severity of chondrocyte degeneration / necrosis was lower (Figure 12C), the severity of bleeding was lower (Figure 12D), the severity of hemosiderin deposition was lower (Figure 12E), the severity of hematoma was lower (Figure 12F), the severity of osteoclastic bone resorption was lower (Figure 12G), the severity of osteolysis was lower (Figure 12H), the severity of periostitis was lower (Figure 12I), the severity of subchondral bone sclerosis was lower (Figure 12J), the severity of tendon degeneration was lower (Figure 12K), the severity of tendinitis was lower (Figure 12L), and the severity of tenosynovitis was lower (Figure 12M) compared to hemA mice that received the vehicle (0.9% saline).

Claims

1. A pharmaceutical composition comprising an inhibitor of ZPI expression and / or function for use in the prevention or treatment of diseases related to hemostatic disorders.

2. The pharmaceutical composition according to claim 1, wherein the disease relating to the hemostatic disorder is hemophilia.

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

4. An inhibitor of ZPI expression and / or function, conjugated to one or more ligand moieties.

5. The inhibitor according to claim 4, which is an siRNA oligomer.

6. An inhibitor of ZPI expression and / or function, which is an siRNA oligomer.

7. The inhibitor according to claim 6, comprising an siRNA oligomer conjugated to one or more ligand portions.

8. The inhibitor or pharmaceutical composition according to claim 3, 4, 5, or 7, wherein the one or more ligand portions comprise one or more GalNAc ligands or one or more GalNAc ligand derivatives.

9. The inhibitor or pharmaceutical composition according to claim 1, 2, 4, or 6, wherein the target of the inhibitor is a ZPI.

10. The inhibitor is an siRNA oligomer having a first chain and a second chain, i) The first strand of the siRNA has a length in the range of 15 to 30 nucleosides, and / or ii) The second strand of the siRNA has a length in the range of 15 to 30 nucleosides. The inhibitor or pharmaceutical composition according to claim 1, 2, 4, or 6.

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

12. The inhibitor or pharmaceutical composition according to claim 1, 2, 4, or 6, wherein one or more nucleosides of the first chain and / or the second chain are modified to form a modified nucleoside.

13. The inhibitor according to claim 4 or 6, formulated as a pharmaceutical composition having an excipient and / or a carrier.

14. A pharmaceutical composition comprising the inhibitor according to claim 4 or 6, in combination with a pharmaceutically acceptable excipient or carrier.

15. A pharmaceutical composition comprising the inhibitor according to claim 4 or 6, in combination with a pharmaceutically acceptable excipient or carrier, for use in the treatment of diseases relating to hemostatic disorders.

16. The pharmaceutical composition according to claim 15, wherein the disease relating to the hemostatic disorder is hemophilia.

17. Use of a ZPI as a target to identify one or more therapeutic agents for treating diseases related to hemostatic disorders.

18. The use according to claim 17, wherein the disease relating to the hemostatic disorder is hemophilia.

19. A ZPI for use as a biomarker for diseases related to hemostatic disorders.

20. The ZPI for use according to claim 19, wherein the disease relating to the hemostatic disorder is hemophilia.

21. Typically, ZPIs are used in vivo methods to predict susceptibility to hemostatic disorders by monitoring the sequence and / or expression and / or function levels of ZPIs in patient samples.

22. The ZPI for use according to claim 21, wherein the disease relating to the hemostatic disorder is hemophilia.

23. A method for predicting a patient's susceptibility to diseases related to hemostatic disorders, (a) To detect the sequence and / or expression and / or function of ZPI in the sample obtained from the patient, (b) Predict susceptibility to hemostatic disorders based on the sequence and / or expression and / or function of ZPIs in the sample obtained from the patient, A method that includes this.

24. The method according to claim 23, wherein the disease relating to the hemostatic disorder is hemophilia.

Images