Conjugated oligonucleotide compounds, methods of making and uses thereof

Abstract

The present invention relates to novel conjugated oligonucleotide compounds suitable for therapeutic use. In addition, the present invention provides methods of making these compounds, as well as methods of using such compounds for the treatment of various diseases and disorders.

Description

[0001] This application is a divisional application of Chinese patent application No. 2022800120078, filed on January 28, 2022, entitled "Conjugated Oligonucleotide Compound, Preparation Method Thereof and Use". Technical Field

[0002] This invention provides novel conjugated oligonucleotide compounds suitable for therapeutic use. Furthermore, this invention provides methods for preparing these compounds, and methods for using such compounds to treat various diseases and conditions. Background Technology

[0003] Oligonucleotide compounds have important therapeutic applications in medicine. Oligonucleotides can be used to silence genes that cause specific diseases. Gene silencing prevents protein formation by inhibiting translation. Importantly, gene silencers are promising alternatives to traditional small organic compounds that inhibit the function of disease-related proteins. siRNA, antisense RNA, and microRNA are oligonucleotides that prevent protein formation through gene silencing.

[0004] Over the past two decades, numerous modified siRNA compounds have been developed, particularly for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutics for treating a wide range of diseases, including central nervous system disorders, inflammatory diseases, metabolic disorders, tumors, infectious diseases, and eye diseases.

[0005] Effective delivery of oligonucleotides into cells requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One approach to achieving specific targeting is to conjugate a ligand-targeting moiety to the oligonucleotide reagent. The ligand-targeting moiety facilitates the delivery of the oligonucleotide to the desired target site. For example, attaching a ligand-targeting moiety containing a terminal galactose or a derivative thereof to an oligonucleotide facilitates targeting hepatocytes via binding to the desialylate glycoprotein receptor (ASGPR).

[0006] There is a need for novel ligand-conjugated oligonucleotides and their preparation methods. Summary of the Invention

[0007] This invention provides novel ligand-conjugated oligonucleotide compounds, methods for preparing these compounds, and their uses.

[0008] This article provides compounds containing the following structures:

[0009]

[0010] Formula (I)

[0011] in:

[0012] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0013] R2 is selected from the group consisting of: hydrogen, hydroxyl group, -OC. 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogenated, and nitro groups;

[0014] X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur;

[0015] m is an integer from 1 to 6;

[0016] n is an integer from 1 to 10;

[0017] q, r, s, t, v are independent integers from 0 to 4, provided that:

[0018] (i) q and r cannot both be 0 at the same time; and

[0019] (ii) s, t, and v cannot all be 0 at the same time;

[0020] Z stands for oligonucleotide.

[0021] This article provides compounds of formula (II):

[0022]

[0023] Equation (II)

[0024] This article provides compounds of formula (III):

[0025]

[0026] Equation (III)

[0027] This document provides a composition comprising a compound of formula (II) as described anywhere herein and a compound of formula (III) as described anywhere herein.

[0028] This article provides compounds of formula (IV):

[0029]

[0030] Formula (IV)

[0031] This article provides compounds of formula (V):

[0032]

[0033] Formula (V)

[0034] This document provides a composition comprising a compound of formula (IV) as described anywhere herein and a compound of formula (V) as described anywhere herein.

[0035] This article provides compounds of formula (VIII):

[0036]

[0037] Formula (VIII)

[0038] This article provides compounds of formula (IX):

[0039]

[0040] Formula (IX)

[0041] This article provides compounds of formula (X):

[0042]

[0043] Formula (X)

[0044] This article provides compounds of formula (XI):

[0045]

[0046] Formula (XI)

[0047] This document provides a composition comprising a compound of formula (VIII) as described anywhere herein and a compound of formula (IX) as described anywhere herein. This document also provides a composition comprising a compound of formula (X) as described anywhere herein and a compound of formula (XI) as described anywhere herein.

[0048] This document provides a method for preparing compounds and / or compositions as described anywhere herein, the method comprising reacting a compound of formula (XII) and a compound of formula (XIII):

[0049]

[0050] Formula (XII)

[0051]

[0052] Formula (XIII)

[0053] in:

[0054] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0055] R2 is selected from the group consisting of: hydrogen, hydroxyl group, -OC.1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogenated, and nitro groups;

[0056] X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur;

[0057] m is an integer from 1 to 6;

[0058] n is an integer from 1 to 10;

[0059] q, r, s, t, v are independent integers from 0 to 4, provided that:

[0060] (i) q and r cannot both be 0 at the same time; and

[0061] (ii) s, t, and v cannot all be 0 at the same time;

[0062] Z is the oligonucleotide moiety;

[0063] And, where appropriate, deprotection of the ligand and / or annealing of the second strand of the oligonucleotide moiety are performed.

[0064] This article provides compounds of formula (XII):

[0065]

[0066] Formula (XII)

[0067] in:

[0068] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0069] R2 is selected from the group consisting of: hydrogen, hydroxyl group, -OC. 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogenated, and nitro groups;

[0070] X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur;

[0071] q, r, s, t, v are independent integers from 0 to 4, provided that:

[0072] (i) q and r cannot both be 0 at the same time; and

[0073] (ii) s, t, and v cannot all be 0 at the same time;

[0074] Z stands for oligonucleotide.

[0075] This article provides compounds of formula (XIIa):

[0076]

[0077] Formula (XIIa)

[0078] This article provides compounds of formula (XIIb):

[0079]

[0080] Formula (XIIb)

[0081] This article provides compounds of formula (XIIc):

[0082]

[0083] Formula (XIIc)

[0084] This article provides compounds of formula (XIId):

[0085]

[0086] Formula (XIId)

[0087] This article provides compounds of formula (XIII):

[0088]

[0089] Formula (XIII)

[0090] in:

[0091] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0092] m is an integer from 1 to 6;

[0093] n is an integer from 1 to 10.

[0094] This article provides compounds of formula (XIIIa):

[0095]

[0096] Equation (XIIIa)

[0097] This article provides compounds of formula (XIIIb):

[0098]

[0099] Equation (XIIIb)

[0100] This article provides compounds of formula (XIV):

[0101]

[0102] Formula (XIV)

[0103] in:

[0104] R1 is selected from the group consisting of: hydrogen, methyl, and ethyl;

[0105] R2 is selected from the group consisting of: hydrogen, hydroxyl group, -OC. 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogenated, and nitro groups;

[0106] X2 is selected from the group consisting of the following: methylene, oxygen, and sulfur;

[0107] s, t, and v are independent integers from 0 to 4, provided that s, t, and v cannot all be 0 at the same time.

[0108] This article provides compounds of formula (XIVa):

[0109]

[0110] Formula (XIVa)

[0111] This article provides compounds of formula (XIVb):

[0112]

[0113] Formula (XIVb)

[0114] This article provides compounds of formula (XV):

[0115]

[0116] Formula (XV)

[0117] in:

[0118] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0119] X1 is selected from the group consisting of the following: methylene, oxygen, and sulfur;

[0120] q and r are independent integers from 0 to 4, provided that q and r cannot both be 0 at the same time;

[0121] Z stands for oligonucleotide.

[0122] This article provides compounds of formula (XVa):

[0123]

[0124] Equation (XVa)

[0125] This article provides compounds of formula (XVb):

[0126]

[0127] Formula (XVb)

[0128] This document provides for the use of the compounds described anywhere herein for the preparation of the compounds described anywhere herein and / or the compositions described anywhere herein.

[0129] This document provides for the use of compounds of formula (XII) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0130] This document provides for the use of the compound of formula (XIII) as described anywhere in this document for the preparation of compounds and / or compositions as described anywhere in this document.

[0131] This document provides for the use of compounds of formula (XIV) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0132] This document provides for the use of compounds of formula (XV) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0133] This document provides for the use of compounds of formula (XIIa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0134] This document provides for the use of compounds of formula (XIIb) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0135] This document provides for the use of compounds of formula (XIIc) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0136] This document provides for the use of compounds of formula (XIId) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0137] This document provides for the use of compounds of formula (XIIIa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0138] This document provides the use of the compound of formula (XIIIb) as described anywhere in this document for the preparation of compounds and / or compositions as described anywhere in this document.

[0139] This document provides for the use of compounds of formula (XIVa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0140] This document provides for the use of compounds of formula (XIVb) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0141] This document provides for the use of compounds of formula (XVa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0142] This document provides the use of compounds of formula (XVb) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0143] This document provides compounds or compositions that are obtained or available by means of methods described anywhere herein.

[0144] This document provides a pharmaceutical composition comprising a compound as described anywhere herein and / or a composition as described anywhere herein, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0145] This document provides for the use of compounds and / or compositions as described anywhere herein for treatment. Attached Figure Description

[0146] Figure 1 Analysis of hsC5 mRNA expression levels in a total of 45 human cancer cell lysates and primary human hepatocyte (PHH) lysates is presented. mRNA expression levels are shown in relative optical units [RLU].

[0147] Figure 2 Analysis of hsHAO1 mRNA expression levels in a total of 45 human cancer cell lysates and primary human hepatocyte (PHH) lysates is presented. mRNA expression levels are shown in relative optical units [RLU].

[0148] Figure 3 Analysis of hsTTR mRNA expression levels in a total of 45 human cancer cell lysates and primary human hepatocyte (PHH) lysates is presented. mRNA expression levels are shown in relative optical units [RLU].

[0149] Figure 4A -D shows the dose-response analysis results of GalNAc-siRNA targeting hsTTR in HepG2 cells in Example 1.

[0150] Figure 5A -D shows the dose-response analysis results of GalNAc-siRNA targeting hsC5 in HepG2 cells in Example 1.

[0151] Figure 6 This paper presents the analysis of hsTTR (top), hsC5 (middle), and hsHAO1 (bottom) mRNA expression levels in all three batches of primary human hepatocytes BHuf16087 (left), CHF2101 (middle), and CyHuf19009 (right) at 0, 24, 48, and 72 hours of culture. mRNA expression levels are shown in relative light units [RLU].

[0152] Figure 7 The analysis shows the expression levels of hsGAPDH (top) and hsAHSA1 (bottom) mRNA in all three batches of primary human hepatocytes BHuf16087 (left), CHF2101 (middle), and CyHuf19009 (right) at 0, 24, 48, and 72 hours of culture. mRNA expression levels are shown in relative light units [RLU].

[0153] Figure 8A -D shows the dose-response analysis results of GalNAc-siRNA targeting hsHAO1 in PHH in Example 1.

[0154] Figure 9A -D shows the dose-response analysis results of GalNAc-siRNA targeting hsC5 in PHH in Example 1.

[0155] Figure 10A -D shows the dose-response analysis results of GalNAc-siRNA targeting hsTTR in PHH in Example 1.

[0156] Figure 11 Pharmacological studies of a single dose of ETX005 in mice. HAO1 mRNA expression relative to the saline control group is shown. Each point represents the mean and standard deviation of 3 mice.

[0157] Figure 12 Single-dose mouse pharmacology of ETX005. Serum glycolic acid concentrations are shown. Each point represents the mean and standard deviation of 3 mice, except for the baseline glycolic acid concentration (day 0) from a group of 5 mice.

[0158] Figure 13Pharmacological studies of a single dose of ETX014 in mice. C5 mRNA expression relative to the saline control group is shown. Each point represents the mean and standard deviation of 3 mice.

[0159] Figure 14 Single-dose mouse pharmacology of ETX0014. Serum C5 concentrations relative to the saline control group are shown. Each point represents the mean and standard deviation of 3 mice.

[0160] Figure 15 Pharmacology of a single dose of ETX023 for NHP. Serum TTR concentrations relative to day 1 of the study are shown. Each point represents the mean and standard deviation of 3 animals.

[0161] Figure 16 Pharmacology of a single dose of ETX019 NHP. Serum TTR concentrations are shown relative to study day 1 and pre-dose. Each point represents the mean and standard deviation of 3 animals. Time points are shown for a maximum of 84 days.

[0162] Figure 17 Pharmacology of a single dose of ETX021 NHP. Serum TTR concentrations are shown relative to study day 1 and pre-dose. Each point represents the mean and standard deviation of 3 animals. Time points are shown for a maximum of 84 days.

[0163] Figure 18a Pharmacology of a single dose of ETX023 for NHP. Serum TTR concentrations are shown relative to study day 1 and pre-dose. Each point represents the mean and standard deviation of 3 animals. Time points are shown for a maximum of 84 days.

[0164] Figure 18b ETX023 consistently suppressed TTR gene expression in the liver following a single 1 mg / kg dose. TTR mRNA levels are shown relative to baseline levels measured before administration. Each point represents the mean and standard deviation of 3 animals. Time points are shown for a maximum of 84 days.

[0165] Figure 18c Body weight of animals administered a single 1 mg / kg dose of ETX023. Each point represents the mean and standard deviation of 3 animals. Time points are shown for a maximum of 84 days.

[0166] Figure 18dSerum ALT concentrations in animals treated with a single 1 mg / kg dose of ETX023. Each point represents the mean and standard deviation of three animals. Shaded areas show the range of values ​​considered normal in the facility used for the study. Dashed lines show the values ​​considered normal for this species (Park et al. 2016 Reference values ​​of clinicalpathology parameter in cynomolgus monkeys used in preclinical studies. LabAnim Res 32:79-86.) showing time points up to 84 days.

[0167] Figure 18e AST concentrations in serum of animals treated with a single 1 mg / kg dose of ETX023. Each point represents the mean and standard deviation of 3 animals. Shaded areas show the range of values ​​considered normal in the facility used for the study. Dashed lines show values ​​considered normal for this species (Park et al. 2016 Reference values ​​of clinicalpathology parameter in cynomolgus monkeys used in preclinical studies. LabAnim Res 32:79-86). Time points up to 84 days are shown.

[0168] Figure 19 Pharmacology of a single dose of ETX025 in NHP. Serum TTR concentrations are shown relative to study day 1 and pre-dose. Each point represents the mean and standard deviation of 3 animals. Time points are shown for a maximum of 84 days.

[0169] Figure 20 The connector and ligand portions of ETX005, 007, 0014, 0016, 0023 and 0025.

[0170] It should also be understood that, where appropriate, although ETX005 as a product includes, for example Figure 20 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX005 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 20 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX005 can be substantially composed of... Figure 20The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX005 may be substantially composed of substantially as follows Figure 20 The described molecular composition has a linker and ligand moiety, but as Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX005 may comprise a mixture of molecules as defined in (a) and / or (b).

[0171] It should also be understood that, where appropriate, although ETX007 as a product includes, for example Figure 20 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX007 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 20 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX007 can be substantially composed of... Figure 20 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX007 can be substantially composed of substantially as follows Figure 20 The described molecular composition has a linker and ligand moiety, but as Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX007 may comprise a mixture of molecules as defined in (a) and / or (b).

[0172] It should also be understood that, where appropriate, although ETX014 as a product includes, for example Figure 20 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX014 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 20 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX014 can be substantially composed of... Figure 20 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX014 can be substantially composed of substantially as follows Figure 20The described molecular composition has a linker and ligand moiety, but as Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX014 may comprise a mixture of molecules as defined in (a) and / or (b).

[0173] It should also be understood that, where appropriate, although ETX016 as a product includes, for example Figure 20 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX016 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 20 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX016 can be substantially composed of... Figure 20 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX016 can be substantially composed of substantially as follows Figure 20 The described molecular composition has a linker and ligand moiety, but as Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX016 may comprise a mixture of molecules as defined in (a) and / or (b).

[0174] It should also be understood that, where appropriate, although ETX023 as a product includes, for example, Figure 20 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX023 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 20 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX023 can be substantially composed of... Figure 20 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX023 can be substantially composed of substantially as follows Figure 20 The described molecular composition has a linker and ligand moiety, but as Figure 20The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX023 may comprise a mixture of molecules as defined in (a) and / or (b).

[0175] It should also be understood that, where appropriate, although ETX025 as a product includes, for example Figure 20 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX025 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 20 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX025 can be substantially composed of... Figure 20 The molecular composition with a linker and ligand moiety specifically depicted in Figure 30, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX025 may consist substantially of a molecular composition with a linker and ligand moiety substantially as depicted in Figure 30, but as Figure 20 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX025 may comprise a mixture of molecules as defined in (a) and / or (b).

[0176] Figure 21 The connector and ligand portions of ETX003, 001, 0010, 0012, 0019 and 0021.

[0177] It should also be understood that, where appropriate, although ETX001 as a product includes, for example Figure 21 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX001 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 21 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX001 can be substantially composed of... Figure 21 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX001 can be substantially composed of substantially as follows Figure 21 The described molecular composition has a linker and ligand moiety, but as Figure 21The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX001 may comprise a mixture of molecules as defined in (a) and / or (b).

[0178] It should also be understood that, where appropriate, although ETX003 as a product includes, for example, Figure 21 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX003 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 21 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX003 can be substantially composed of... Figure 21 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX003 can be substantially composed of substantially as follows Figure 21 The described molecular composition has a linker and ligand moiety, but as Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX003 may comprise a mixture of molecules as defined in (a) and / or (b).

[0179] It should also be understood that, where appropriate, although ETX010 as a product includes, for example Figure 21 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX010 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 21 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX010 can be substantially composed of... Figure 21 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX010 can be substantially composed of substantially as follows Figure 21 The described molecular composition has a linker and ligand moiety, but as Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX010 may comprise a mixture of molecules as defined in (a) and / or (b).

[0180] It should also be understood that, where appropriate, although ETX012 as a product includes, for example, Figure 21 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX012 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 21 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX012 can be substantially composed of... Figure 21 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX012 can be substantially composed of substantially as follows Figure 21 The described molecular composition has a linker and ligand moiety, but as Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX012 may comprise a mixture of molecules as defined in (a) and / or (b).

[0181] It should also be understood that, where appropriate, although ETX019 as a product includes, for example Figure 21 The molecule specifically described herein is based on a linker and ligand moiety attached to an oligonucleotide moiety also described herein, but the ETX019 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand moiety are substantially as described herein. Figure 21 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX019 can be substantially composed of... Figure 21 The molecular composition with a linker and ligand moiety specifically depicted in Figure 30, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX019 may consist essentially of a molecular composition with a linker and ligand moiety as depicted in Figure 30, but as Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX019 may comprise a mixture of molecules as defined in (a) and / or (b).

[0182] It should also be understood that, where appropriate, although ETX021 as a product includes, for example Figure 21The molecule specifically described herein is based on a linker and ligand motif, which is attached to an oligonucleotide motif also described herein. However, the ETX021 product may alternatively further comprise or consist substantially of the following molecules: in which the linker and ligand motifs are substantially as described herein. Figure 21 As depicted, the molecule is attached to an oligonucleotide moiety, but as... Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution. In this way, (a) ETX021 can be substantially composed of... Figure 21 The molecular composition specifically described in the text, having a linker and ligand moiety, wherein an F substituent is present on the cyclooctyl ring; or (b) ETX021 can be substantially composed of substantially as follows Figure 21 The described molecular composition has a linker and ligand moiety, but as Figure 21 The F substituent on the cyclooctyl ring shown is replaced by a substituent (such as an OH substituent) resulting from hydrolytic substitution, or (c) ETX021 may comprise a mixture of molecules as defined in (a) and / or (b).

[0183] Figure 22 Total bilirubin concentrations in the serum of animals treated with a single 1 mg / kg dose of ETX023. Each point represents the mean and standard deviation of three animals. The dashed line shows the range of values ​​considered normal for this species (Park et al. 2016 Reference values ​​of clinical pathology parameter in cynomolgus monkeys used in preclinical studies. Lab Anim Res 32:79-86.).

[0184] Figure 23 Blood urea nitrogen (BUN) concentrations in animals treated with a single 1 mg / kg dose of ETX023. Each point represents the mean and standard deviation of three animals. The dashed line shows the range of values ​​considered normal for this species (Park et al. 2016 Reference values ​​of clinical pathology parameter in cynomolgus monkeys used in preclinical studies. Lab Anim Res 32:79-86.).

[0185] Figure 24Creatinine (CREA) concentrations in animals treated with a single 1 mg / kg dose of ETX023. Each point represents the mean and standard deviation of three animals. The dashed line shows the range of values ​​considered normal for this species (Park et al. 2016 Reference values ​​of clinical pathology parameter in cynomolgus monkeys used in preclinical studies. Lab Anim Res 32:79-86.).

[0186] Figures 25-27 are described in more detail below.

[0187] Figure 28 The details of equations I to XV are shown below.

[0188] Figure 29a The basic nucleotide sequences of the sense (SS) and antisense (AS) strands of the construct ETX001 as described herein are shown. For ETX001, the galnac linker is attached to the 5' end region of the sense strand in use. Figure 29a (Not depicted in the text). For ETX001, the galnac connector is attached and as... Figure 21 As shown. Mentioned in subsequent paragraphs. Figure 29a It refers to the sequence, construct design, and decoration mode of ETX001.

[0189] like Figure 29a The iaia shown in the 3' end region of the sense strand indicates (i) two abasic nucleotides provided as the penultimate and terminal nucleotides in the 3' end region of the sense strand, (ii) a 3'-3' reverse link provided between the penultimate nucleotide of the sense strand (i.e., A at position 21 of the sense strand, where position 1 is the terminal 5' nucleotide of the sense strand, i.e., G at the end of the 5' end region of the sense strand) and the adjacent penultimate abasic residue, and (iii) the link between the terminal and the penultimate abasic nucleotide is 5'-3' when read toward the 3' end region containing the terminal and penultimate abasic nucleotides.

[0190] for Figure 29a The sense strand, when read from position 1 of the sense strand (the terminal 5' nucleotide of the sense strand, i.e., the terminal G in the 5' end region of the sense strand), then: (i) the nucleotides at positions 1 to 6, 8 and 12 to 21 have a 2' O-methyl modified sugar, (ii) the nucleotides at positions 7 and 9 to 11 have a 2' F modified sugar, and (iii) the non-base nucleotides have a sugar with H at positions 1 and 2.

[0191] for Figure 29aThe antisense strand, when read from position 1 of the antisense strand (which is the terminal 5' nucleotide of the antisense strand, i.e., the terminal U in the 5' end region of the antisense strand), then: (i) the nucleotides at positions 1, 3 to 5, 7, 10 to 13, 15, 17 to 23 have a 2'O-methyl modified sugar, and (ii) the nucleotides at positions 2, 6, 8, 9, 14, 16 have a 2'F modified sugar.

[0192] As described in this article, ETX003 has the same... Figure 29a The nucleotide exhibits the same basic sequence and galnac linker and attachment as described for ETX001, but lacks the terminal iaia motif and has a completely alternating 2'O-methyl / 2'F modification pattern on the sugar of the nucleotide. For the sense strand, the completely alternating modification pattern begins at position 1 of the 2'F modification in the 5' end region of the sense strand. For the antisense strand, the completely alternating modification pattern begins at position 1 of the 2'O-methyl modification in the 5' end region of the antisense strand.

[0193] Figure 29b The basic nucleotide sequences of the sense (SS) and antisense (AS) strands of the construct ETX005 as described herein are shown. For ETX005, the galnac linker is attached to the 3' end region of the sense strand in use. Figure 29b (Not depicted in the text). For ETX005, the galnac connector is attached and as... Figure 20 As shown. Mentioned in subsequent paragraphs. Figure 29b It refers to the sequence, construct design, and decoration mode of ETX005.

[0194] like Figure 29b The iaia shown in the 5' end region of the sense strand indicates (i) two abasic nucleotides provided as the penultimate and terminal nucleotides in the 5' end region of the sense strand, (ii) a 5'-5' reverse link provided between the penultimate nucleotide of the sense strand (i.e., the G at position 1 of the sense strand, which is not included in the iaia motif of the 5' end region of the sense strand in the nucleotide position numbering on the sense strand) and the adjacent penultimate abasic residue, and (iii) the link between the terminal and the penultimate abasic nucleotide is 3'-5' when read toward the 5' end region containing the terminal and penultimate abasic nucleotides.

[0195] for Figure 29bThe sense strand, when read from position 1 of the sense strand (the terminal 5' nucleotide of the sense strand, i.e., the terminal G in the 5' end region of the sense strand, iaia motif not included in the 5' end region of the sense strand in the nucleotide position numbering of the sense strand), then: (i) the nucleotides at positions 1 to 6, 8 and 12 to 21 have a 2'O-methyl modified sugar, (ii) the nucleotides at positions 7 and 9 to 11 have a 2'F modified sugar, and (iii) the non-base nucleotides have a sugar with H at positions 1 and 2.

[0196] for Figure 29b The antisense strand, when read from position 1 of the antisense strand (which is the terminal 5' nucleotide of the antisense strand, i.e., the terminal U in the 5' end region of the antisense strand), then: (i) the nucleotides at positions 1, 3 to 5, 7, 10 to 13, 15, 17 to 23 have a 2'O-methyl modified sugar, and (ii) the nucleotides at positions 2, 6, 8, 9, 14, 16 have a 2'F modified sugar.

[0197] As described in this article, ETX007 has the same... Figure 29b The nucleotide exhibits the same basic sequence and galnac linker and attachment as described for ETX005, but lacks the terminal iaia motif and has a completely alternating 2'O-methyl / 2'F modification pattern on the sugar of the nucleotide. For the sense strand, the completely alternating modification pattern begins at position 1 of the 2'F modification in the 5' end region of the sense strand. For the antisense strand, the completely alternating modification pattern begins at position 1 of the 2'O-methyl modification in the 5' end region of the antisense strand.

[0198] Figure 30a The basic nucleotide sequences of the sense (SS) and antisense (AS) strands of the construct ETX010 as described herein are shown. For ETX010, the galnac linker is attached to the 5' end region of the sense strand in use. Figure 30a (Not depicted in the text). For ETX010, the galnac connector is attached and as... Figure 21 As shown. Mentioned in subsequent paragraphs. Figure 30a It refers to the sequence, architecture design, and decoration mode of ETX010.

[0199] like Figure 30aThe iaia shown in the 3' end region of the sense strand indicates (i) two abasic nucleotides provided as the penultimate and terminal nucleotides in the 3' end region of the sense strand, (ii) a 3'-3' reverse link provided between the penultimate nucleotide of the sense strand (i.e., A at position 21 of the sense strand, where position 1 is the terminal 5' nucleotide of the sense strand, i.e., the terminal A in the 5' end region of the sense strand) and the adjacent penultimate abasic residue, and (iii) the link between the terminal and the penultimate abasic nucleotide is 5'-3' when read toward the 3' end region containing the terminal and penultimate abasic nucleotides.

[0200] for Figure 30a The sense strand, when read from position 1 of the sense strand (the terminal 5' nucleotide of the sense strand, i.e., the terminal A of the 5' end region of the sense strand), then: (i) the nucleotides at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, 19 to 21 have a 2'O-methyl modified sugar, (ii) the nucleotides at positions 3, 5, 7, 9 to 11, 13, 16, 18 have a 2'F modified sugar, and (iii) the non-base nucleotides have a sugar with H at positions 1 and 2.

[0201] for Figure 30a The antisense strand, when read from position 1 of the antisense strand (which is the terminal 5' nucleotide of the antisense strand, i.e., the terminal U in the 5' end region of the antisense strand), then: (i) the nucleotides at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, 19 to 23 have a 2'O-methyl modified sugar, (ii) the nucleotides at positions 2, 3, 5, 8, 10, 14, 16, 18 have a 2'F modified sugar, and (iii) the penultimate and terminal T nucleotides at positions 24 and 25 in the 3' end region of the antisense strand have a sugar with an H at position 2.

[0202] As described in this article, ETX012 has the same... Figure 30a The nucleotides described for ETX010 have the same basic sequence and galnac linker and attachment, but lack the terminal iaia motif and have a completely alternating 2'O-methyl / 2'F modification pattern on the sugar of the nucleotide (except for the terminal T nucleotide with H at position 2). For the sense strand, the completely alternating modification pattern begins with the 2'F modification at position 1 in the 5' end region of the sense strand. For the antisense strand, the completely alternating modification pattern begins with the 2'O-methyl modification at position 1 in the 5' end region of the antisense strand.

[0203] Figure 30b The basic nucleotide sequences of the sense (SS) and antisense (AS) strands of the construct ETX014 as described herein are shown. For ETX014, the galnac linker is attached to the 3' end region of the sense strand in use. Figure 30b(Not depicted in the text). For ETX014, the galnac connector is attached and as... Figure 20 As shown. Mentioned in subsequent paragraphs. Figure 30b It refers to the sequence, construct design, and decoration mode of ETX014.

[0204] like Figure 30b The iaia shown in the 5' end region of the sense strand indicates (i) two abasic nucleotides provided as the penultimate and terminal nucleotides in the 5' end region of the sense strand, (ii) a 5'-5' reverse link provided between the penultimate nucleotide of the sense strand (i.e., A at position 1 of the sense strand, which is not included in the iaia motif of the 5' end region of the sense strand in the nucleotide position numbering on the sense strand) and the adjacent penultimate abasic residue, and (iii) the link between the terminal and the penultimate abasic nucleotide is 3'-5' when read toward the 5' end region containing the terminal and penultimate abasic nucleotides.

[0205] for Figure 30b The sense strand, when read from position 1 of the sense strand (the terminal 5' nucleotide of the sense strand, i.e., the terminal A in the 5' end region of the sense strand, iaia motif not included in the 5' end region of the sense strand in the nucleotide position numbering of the sense strand), then: (i) the nucleotides at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, 19 to 21 have a 2'O-methyl modified sugar, (ii) the nucleotides at positions 3, 5, 7, 9 to 11, 13, 16, 18 have a 2'F modified sugar, and (iii) the non-base nucleotides have a sugar with H at positions 1 and 2.

[0206] for Figure 30b The antisense strand, when read from position 1 of the antisense strand (which is the terminal 5' nucleotide of the antisense strand, i.e., the terminal U in the 5' end region of the antisense strand), then: (i) the nucleotides at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, 19 to 23 have a 2'O-methyl modified sugar, (ii) the nucleotides at positions 2, 3, 5, 8, 10, 14, 16, 18 have a 2'F modified sugar, and (iii) the penultimate and terminal T nucleotides at positions 24 and 25 in the 3' end region of the antisense strand have a sugar with an H at position 2.

[0207] As described in this article, ETX016 has the same... Figure 30bThe nucleotides described for ETX014 have the same basic sequence and galnac linker and attachment, but lack the terminal iaia motif and have a completely alternating 2'O-methyl / 2'F modification pattern on the sugar of the nucleotide (except for the terminal T nucleotide with H at position 2). For the sense strand, the completely alternating modification pattern begins with the 2'F modification at position 1 in the 5' end region of the sense strand. For the antisense strand, the completely alternating modification pattern begins with the 2'O-methyl modification at position 1 in the 5' end region of the antisense strand.

[0208] Figure 31a The basic nucleotide sequences of the sense (SS) and antisense (AS) strands of the construct ETX019 as described herein are shown. For ETX019, the galnac linker is attached to the 5' end region of the sense strand in use. Figure 31a (Not depicted in the text). For ETX019, the galnac connector is attached and as... Figure 21 As shown. Mentioned in subsequent paragraphs. Figure 31a It refers to the sequence, construct design, and decoration mode of ETX019.

[0209] like Figure 31a The iaia shown in the 3' end region of the sense strand indicates (i) two abasic nucleotides provided as the penultimate and terminal nucleotides in the 3' end region of the sense strand, (ii) a 3'-3' reverse link provided between the penultimate nucleotide of the sense strand (i.e., A at position 21 of the sense strand, where position 1 is the terminal 5' nucleotide of the sense strand, i.e., the terminal U in the 5' end region of the sense strand) and the adjacent penultimate abasic residue, and (iii) the link between the terminal and the penultimate abasic nucleotide is 5'-3' when read toward the 3' end region containing the terminal and penultimate abasic nucleotides.

[0210] for Figure 31a The sense chain, when read from position 1 of the sense chain (the terminal 5' nucleotide of the sense chain, i.e., the terminal U in the 5' end region of the sense chain), then: (i) the nucleotides at positions 1 to 6, 8 and 12 to 21 have a 2'O-methyl modified sugar, (ii) the nucleotides at positions 7 and 9 to 11 have a 2'F modified sugar, and (iii) the non-base nucleotides have a sugar with H at positions 1 and 2.

[0211] for Figure 31a The antisense strand, when read from position 1 of the antisense strand (which is the terminal 5' nucleotide of the antisense strand, i.e., the terminal U in the 5' end region of the antisense strand), then: (i) the nucleotides at positions 1, 3 to 5, 7, 8, 10 to 13, 15, 17 to 23 have a 2'O-methyl modified sugar, and (ii) the nucleotides at positions 2, 6, 9, 14, 16 have a 2'F modified sugar.

[0212] As described in this article, ETX021 has the same... Figure 31a The nucleotide has the same basic sequence and galnac linker and attachment as described for ETX019, but lacks the terminal iaia motif and has a completely alternating 2'O-methyl / 2'F modification pattern on the sugar of the nucleotide. For the sense strand, the completely alternating modification pattern begins at position 1 of the 2'F modification in the 5' end region of the sense strand. For the antisense strand, the completely alternating modification pattern begins at position 1 of the 5' end region of the antisense strand.

[0213] Figure 31b The basic nucleotide sequences of the sense (SS) and antisense (AS) strands of the construct ETX023 as described herein are shown. For ETX023, the galnac linker is attached to the 3' end region of the sense strand in use. Figure 31b (Not depicted in the text). For ETX023, the galnac connector is attached and as... Figure 20 As shown. Mentioned in subsequent paragraphs. Figure 31b It refers to the sequence, construct design, and decoration mode of ETX023.

[0214] like Figure 31b The iaia shown in the 5' end region of the sense strand indicates (i) two abasic nucleotides provided as the penultimate and terminal nucleotides in the 5' end region of the sense strand, (ii) a 5'-5' reverse link provided between the penultimate nucleotide of the sense strand (i.e., the U at position 1 of the sense strand, which is not included in the iaia motif of the 5' end region of the sense strand in the nucleotide position numbering on the sense strand) and the adjacent penultimate abasic residue, and (iii) the link between the terminal and the penultimate abasic nucleotide is 3'-5' when read toward the 5' end region containing the terminal and penultimate abasic nucleotides.

[0215] for Figure 31b The sense strand, when read from position 1 of the sense strand (the terminal 5' nucleotide of the sense strand, i.e., the terminal U in the 5' end region of the sense strand, iaia motif not included in the 5' end region of the sense strand in the nucleotide position numbering of the sense strand), then: (i) the nucleotides at positions 1 to 6, 8 and 12 to 21 have a 2'O-methyl modified sugar, (ii) the nucleotides at positions 7 and 9 to 11 have a 2'F modified sugar, and (iii) the non-base nucleotides have a sugar with H at positions 1 and 2.

[0216] for Figure 31bThe antisense strand, when read from position 1 of the antisense strand (which is the terminal 5' nucleotide of the antisense strand, i.e., the terminal U in the 5' end region of the antisense strand), then: (i) the nucleotides at positions 1, 3 to 5, 7, 8, 10 to 13, 15, 17 to 23 have a 2'O-methyl modified sugar, and (ii) the nucleotides at positions 2, 6, 9, 14, 16 have a 2'F modified sugar.

[0217] As described in this article, ETX025 has the same... Figure 31b The nucleotide has the same basic sequence and galnac linker and attachment as described for ETX023, but lacks the terminal iaia motif and has a completely alternating 2'O-methyl / 2'F modification pattern on the sugar of the nucleotide. For the sense strand, the completely alternating modification pattern begins at position 1 of the 2'F modification in the 5' end region of the sense strand. For the antisense strand, the completely alternating modification pattern begins at position 1 of the 5' end region of the antisense strand. Detailed Implementation

[0218] This invention provides novel ligand-conjugated oligonucleotide compounds, methods for preparing these compounds, and their uses.

[0219] As disclosed herein, the compounds of the present invention comprise an oligonucleotide moiety and / or a linker and / or a ligand moiety, or portions thereof. Preferably, the compounds of the present invention comprise an oligonucleotide moiety, a linker, and a ligand moiety. These moieties may be covalently linked such that the oligonucleotide moiety is covalently linked to the ligand moiety via the linker.

[0220] It should be understood that the compounds of the present invention can be combined with any oligonucleotide moiety as described anywhere herein, and / or any linker as described anywhere herein, and / or any ligand moiety as described anywhere herein.

[0221] The exemplary compounds of the present invention comprise the following general structures:

[0222]

[0223] Formula (I)

[0224] in:

[0225] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0226] R2 is selected from the group consisting of: hydrogen, hydroxyl group, -OC. 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogenated, and nitro groups;

[0227] X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur;

[0228] m is an integer from 1 to 6;

[0229] n is an integer from 1 to 10;

[0230] q, r, s, t, v are independent integers from 0 to 4, provided that:

[0231] (i) q and r cannot both be 0 at the same time; and

[0232] (ii) s, t, and v cannot all be 0 at the same time;

[0233] Z stands for oligonucleotide.

[0234] 1. Ligand component

[0235] The exemplary compounds of the present invention comprise a 'ligand moiety' as depicted in formula (I).

[0236] In some embodiments, the ligand portion as depicted in formula (I) comprises one or more ligands.

[0237] In some embodiments, the ligand portion as depicted in formula (I) comprises one or more carbohydrate ligands.

[0238] In some embodiments, one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and / or polysaccharides.

[0239] In some embodiments, one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0240] In some embodiments, one or more carbohydrates contain one or more N-acetyl-galactosamine moieties.

[0241] In some embodiments, the compound, as described anywhere herein, comprises two or three N-acetylgalactosamine moieties.

[0242] In some embodiments, one or more ligands are attached in a linear or branched configuration, for example, each configuration is attached to a branch point in the overall junction.

[0243] Exemplary linear or branched configurations of the ligand portion can be described using the nomenclature further explained in Sections 2, 3 and 4 below.

[0244] Exemplary linear configurations such as Figure 32 As shown, (a) and / or (b) can typically represent a linking bond or group, such as a phosphoric acid or thiophosphate group.

[0245] Exemplary branch configurations are as follows Figure 33 As shown.

[0246] In some embodiments, one or more ligands are attached in a biantennary or triantennary branching configuration. Typically, a triantennary branching configuration, such as the N-acetylgalactosamine triantennary branching configuration, is preferred.

[0247] 2. Connector

[0248] The exemplary compounds of the present invention include a 'connector portion' as depicted in formula (I), which is part of the overall 'connector'.

[0249] As will be further understood in the art, the exemplary compounds of the present invention comprise a general connector located between the oligonucleotide portion and the ligand portion of these compounds. The general connector thus 'connects' the oligonucleotide portion and the ligand portion to each other.

[0250] A general linker is typically conceived theoretically as comprising one or more linker structural units. For example, there exists a linker portion described as a 'linker portion' as shown in Formula (I), positioned adjacent to a ligand portion and typically attaching the ligand portion directly or indirectly to the oligonucleotide portion via a branching point. The linker portion depicted in Formula (I) may also be referred to as a 'ligand arm' of the general linker. Further linker portions may exist, but are not always present, between the oligonucleotide portion and the branching point; these linker portions are typically referred to as 'tethering portions' of the general linker, which 'tether' the oligonucleotide portion to the remainder of the conjugated compound. Such 'ligand arms' and / or 'linker portions' and / or 'tethering portions' can be conceived by referring to the linear and / or branched configurations listed above.

[0251] As can be seen from the claims and the remainder of the patent specification, the scope of the invention extends to linear or branched configurations and is not limited to the number of individual ligands that may be present. Furthermore, the visitor will recognize that, based on prior art and the expertise of oligonucleotide chemists, numerous structures exist that can be used as linker portions.

[0252] The remaining portions of the general connector (excluding the connector portion) listed in the claims and the remainder of the patent specification are indicated by their chemical composition in formula (I), which the inventors consider to be particularly unique to the present invention. However, in more general terms, these chemical components may be described as 'tether portions' as described above, where 'tether portions' are the portions of the general connector containing the groups between Z (i.e., oligonucleotide portions) and the connector portions as described in formula (I).

[0253] 2.1 Chain Section

[0254] Regarding formula (I), the 'linkage portion' includes the atomic group between Z (i.e., the oligonucleotide portion) and the linker portion.

[0255] In some embodiments, R1 is hydrogen each time it appears. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl.

[0256] In some embodiments, R2 is a hydroxyl group. In some embodiments, R2 is a halogenated group. In some embodiments, R2 is a fluorine group. In some embodiments, R2 is a chlorine group. In some embodiments, R2 is a bromine group. In some embodiments, R2 is an iodine group. In some embodiments, R2 is a nitro group.

[0257] In some embodiments, X1 is methylene. In some embodiments, X1 is oxygen. In some embodiments, X1 is sulfur.

[0258] In some embodiments, X2 is methylene. In some embodiments, X2 is oxygen. In some embodiments, X2 is sulfur.

[0259] In some embodiments, m = 3.

[0260] In some embodiments, n = 6.

[0261] In some embodiments, X1 is oxygen and X2 is methylene. In some embodiments, both X1 and X2 are methylene.

[0262] 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.

[0263] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is fluorine, X2 is methylene, v = 1, t = 1, s = 1, X1 is methylene, q = 1, and r = 2.

[0264] Therefore, in some embodiments, the exemplary compounds of the present invention comprise the following structures:

[0265]

[0266] Formula (IV)

[0267] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is fluorine, X2 is methylene, v = 1, t = 1, s = 1, X1 is oxygen, q = 1, and r = 2.

[0268] Therefore, in some embodiments, the exemplary compounds of the present invention comprise the following structures:

[0269]

[0270] Equation (II)

[0271] 2.1.1 Alternative Linkages

[0272] During the synthesis of the compounds of this invention, alternative chain portion structures may emerge. In some embodiments, the alternative chain portion has one or more atomic variations in the chain portion of the general connector compared to the chain portion described anywhere herein.

[0273] In some embodiments, the alternative chain portion is a compound of formula (I) as described anywhere herein, wherein R2 is a hydroxyl group.

[0274] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is hydroxyl, X2 is methylene, v = 1, t = 1, s = 1, X1 is methylene, q = 1, and r = 2.

[0275] Therefore, in some embodiments, the compounds of the present invention comprise the following structures:

[0276]

[0277] Formula (V)

[0278] In some embodiments, R1 is hydrogen each time it appears, n = 6, m = 3, R2 is hydroxyl, X2 is methylene, v = 1, t = 1, s = 1, X1 is oxygen, q = 1, and r = 2.

[0279] Therefore, in some embodiments, the compounds of the present invention comprise the following structures:

[0280]

[0281] Equation (III)

[0282] 2.2 Connector section

[0283] Regarding formula (I), the 'connector portion' described in formula (I) comprises an atomic group located between the chain portion as described anywhere in this document and the ligand portion as described anywhere in this document.

[0284] In some embodiments:

[0285]

[0286] As described anywhere in this document, formula (I) depicts any of formulas (VIa), (VIb), or (VIc), with formula (VIa) being preferred:

[0287]

[0288] Formula (VIa)

[0289] in:

[0290] A I It is a hydrogen or a suitable hydroxyl protecting group;

[0291] a is an integer of 2 or 3; and

[0292] b is an integer from 2 to 5; or

[0293]

[0294] Formula (VIb)

[0295] in:

[0296] A I It is a hydrogen or a suitable hydroxyl protecting group;

[0297] a is an integer of 2 or 3; and

[0298] c and d are independent integers from 1 to 6; or

[0299]

[0300] Formula (VIc)

[0301] in:

[0302] A I It is a hydrogen or a suitable hydroxyl protecting group;

[0303] a is an integer of 2 or 3; and

[0304] e is an integer from 2 to 10.

[0305] In some embodiments, the portion depicted in formula (I) as described anywhere herein:

[0306]

[0307] This is formula (VIa):

[0308]

[0309] Formula (VIa)

[0310] in:

[0311] AI It is a hydrogen or a suitable hydroxyl protecting group;

[0312] a is 3; and

[0313] b is an integer 3.

[0314] In some embodiments, the portion depicted in formula (I) as described anywhere herein:

[0315]

[0316] Equation (VII):

[0317]

[0318] Equation (VII)

[0319] in:

[0320] A I It is hydrogen;

[0321] a is an integer of 2 or 3, preferably 3.

[0322] 3. Oligonucleotide portion

[0323] The exemplary compounds of the present invention comprise an oligonucleotide moiety, which is described as 'Z' in formula (I).

[0324] In some embodiments, Z is:

[0325]

[0326] in:

[0327] Z1, Z2, Z3, and Z4 are independently oxygen or sulfur each time they appear; and

[0328] One of the bonds between P and Z2, and between P and Z3, is a single bond, and the other bond is a double bond.

[0329] In some embodiments, oligonucleotides are RNA compounds capable of regulating the expression of target genes. In some embodiments, oligonucleotides are RNA compounds capable of inhibiting the expression of target genes.

[0330] In some embodiments, the RNA compound comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first and second strands has a 5' end and a 3' end.

[0331] In some embodiments, the first strand is at least 80% complementary to the RNA sequence of the target gene, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, such as being 100% complementary in length of the first strand.

[0332] In some embodiments, the RNA compound is attached to an adjacent phosphate group at the 5' end of its second strand.

[0333] In some embodiments, the RNA compound is attached to an adjacent phosphate group at the 3' end of its second strand.

[0334] It should be understood that when an RNA compound is attached to the 5' end of the second strand, the phosphate group that connects the oligonucleotide to the linker portion (i.e., the 'P' attached to Z1, Z2, Z3, and Z4) is a naturally occurring phosphate group from the 5' terminal ribose of the oligonucleotide.

[0335] It should be understood that when an RNA compound is attached to the 3' end of the second strand, the phosphate group that links the oligonucleotide to the linker portion (i.e., the 'P' linked to Z1, Z2, Z3, and Z4) is engineered onto the 3' terminal ribose of the oligonucleotide to replace the naturally present hydroxyl group at position 3'.

[0336] In some embodiments, the oligonucleotide comprises an RNA duplex that further comprises one or more riboses modified at position 2'. In some embodiments, the RNA duplex comprises multiple riboses modified at position 2'. In some embodiments, the modification is selected from 2'-O-methyl, 2'-deoxy-fluorine, and 2'-deoxy.

[0337] In some embodiments, the oligonucleotide further includes one or more degradation-protecting portions at one or more ends. In some embodiments, the one or more degradation-protecting portions are not present at the ends of the ligand-carrying portions of the oligonucleotide chain. In some embodiments, the one or more degradation-protecting portions are not present at the ends of the oligonucleotide chain adjacent to the remainder of the compound as shown in formulas (I), (VII), (IX), (X), or (XI). In some embodiments, the one or more degradation-protecting portions are selected from phosphate-thionucleotide inter-linked bonds, phosphate-dithionucleotide inter-linked bonds, and inverse abase-free nucleotides, wherein the inverse abase-free nucleotide is present at the distal end of the ligand-carrying portion of the chain.

[0338] 4. Exemplary Compounds

[0339] The compounds of the present invention incorporate any oligonucleotide moiety as described anywhere herein, any linker as described anywhere herein, and / or any ligand moiety as described anywhere herein, or portions thereof.

[0340] In some embodiments, the compound comprises formula (VIII):

[0341]

[0342] In some embodiments, the compound comprises formula (IX):

[0343]

[0344] In some embodiments, the compound comprises formula (X):

[0345]

[0346] In some embodiments, the compound comprises formula (XI):

[0347]

[0348] 4.1 Intermediate Compounds

[0349] The compounds of the present invention also include intermediate compounds produced or used during the production methods of the present invention as described anywhere herein, which are used to produce the compounds as described anywhere herein.

[0350] Therefore, in some embodiments, the compound comprises formula (XII):

[0351]

[0352] Formula (XII)

[0353] in:

[0354] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0355] R2 is selected from the group consisting of: hydroxyl, halogen, and nitro;

[0356] X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur;

[0357] q, r, s, t, v are independent integers from 0 to 4, provided that:

[0358] (i) q and r cannot both be 0 at the same time; and

[0359] (ii) s, t, and v cannot all be 0 at the same time;

[0360] Z stands for oligonucleotide.

[0361] In some embodiments, the compound comprises formula (XIIa):

[0362]

[0363] Formula (XIIa)

[0364] In some embodiments, the compound comprises formula (XIIb):

[0365]

[0366] Formula (XIIb)

[0367] In some embodiments, the compound comprises formula (XIIc):

[0368]

[0369] Formula (XIIc)

[0370] In some embodiments, the compound comprises formula (XIId):

[0371]

[0372] Formula (XIId)

[0373] In some embodiments, the compound comprises formula (XIII):

[0374]

[0375] Formula (XIII)

[0376] in:

[0377] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0378] m is an integer from 1 to 6;

[0379] n is an integer from 1 to 10.

[0380] In some embodiments, the compound comprises formula (XIIIa):

[0381]

[0382] Equation (XIIIa)

[0383] In some embodiments, the compound comprises formula (XIIIb):

[0384]

[0385] Equation (XIIIb)

[0386] In some embodiments, the compound comprises formula (XIV):

[0387]

[0388] Formula (XIV)

[0389] in:

[0390] R1 is selected from the group consisting of: hydrogen, methyl, and ethyl;

[0391] R2 is selected from the group consisting of: hydroxyl, halogen, and nitro;

[0392] X2 is selected from the group consisting of the following: methylene, oxygen, and sulfur;

[0393] s, t, and v are independent integers from 0 to 4, provided that s, t, and v cannot all be 0 at the same time.

[0394] In some embodiments, the compound comprises formula (XIVa):

[0395]

[0396] Formula (XIVa)

[0397] In some embodiments, the compound comprises formula (XIVb):

[0398]

[0399] Formula (XIVb)

[0400] In some embodiments, the compound comprises formula (XV):

[0401]

[0402] Formula (XV)

[0403] in:

[0404] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0405] X1 is selected from the group consisting of the following: methylene, oxygen, and sulfur;

[0406] q and r are independent integers from 0 to 4, provided that q and r cannot both be 0 at the same time;

[0407] Z stands for oligonucleotide.

[0408] In some embodiments, the compound comprises the formula (XVa):

[0409]

[0410] Equation (XVa)

[0411] In some embodiments, the compound comprises the formula (XVb):

[0412]

[0413] Formula (XVb)

[0414] 5. Composition

[0415] This invention relates to compositions comprising combinations of the compounds of this invention.

[0416] In some embodiments, the combination comprises a compound containing alternative portions as described anywhere herein.

[0417] In some embodiments, compounds in compositions comprising alternative chain portions as described anywhere herein are present in an amount of 10% by weight or less of the composition.

[0418] In some embodiments, compounds in a composition comprising alternative chain portions as described anywhere herein are present in an amount ranging from 10% to 15% by weight of the composition.

[0419] In some embodiments, the composition comprises a compound of formula (IV) as described anywhere herein and a compound of formula (V) as described anywhere herein. In some embodiments, the compound of formula (V) as described anywhere herein is present in an amount ranging from 10% to 15% by weight of the composition.

[0420] In some embodiments, the composition comprises a compound of formula (X) as described anywhere herein and a compound of formula (XI) as described anywhere herein. In some embodiments, the compound of formula (XI) as described anywhere herein is present in an amount ranging from 10% to 15% by weight of the composition.

[0421] In some embodiments, the composition comprises a compound of formula (II) as described anywhere herein and a compound of formula (III) as described anywhere herein. In some embodiments, the compound of formula (III) as described anywhere herein is present in an amount ranging from 10% to 15% by weight of the composition.

[0422] In some embodiments, the composition comprises a compound of formula (VIII) as described anywhere herein and a compound of formula (IX) as described anywhere herein.

[0423] In some embodiments, the compound of formula (IX) as described anywhere herein is present in an amount ranging from 10% to 15% by weight of the composition.

[0424] 6. Production methods

[0425] The present invention further provides a method for preparing compounds as described anywhere herein. The present invention further provides a method for preparing compositions as described anywhere herein.

[0426] In some embodiments, the method includes reacting the compound of formula (XII) and the compound of formula (XIII):

[0427]

[0428] Formula (XII)

[0429]

[0430] Formula (XIII)

[0431] in:

[0432] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0433] R2 is selected from the group consisting of: hydrogen, hydroxyl group, -OC. 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogenated, and nitro groups;

[0434] X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur;

[0435] m is an integer from 1 to 6;

[0436] n is an integer from 1 to 10;

[0437] q, r, s, t, v are independent integers from 0 to 4, provided that:

[0438] (i) q and r cannot both be 0 at the same time; and

[0439] (ii) s, t, and v cannot all be 0 at the same time;

[0440] Z is the oligonucleotide moiety;

[0441] And, where appropriate, deprotection of the ligand and / or annealing of the second strand of the oligonucleotide moiety are performed.

[0442] In some embodiments, the compound of formula (XII) is of formula (XIIa):

[0443]

[0444] Formula (XIIa)

[0445] And the compound of formula (XIII) is of formula (XIIIa):

[0446]

[0447] Equation (XIIIa)

[0448] The oligonucleotide comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, wherein each of the first strand and the second strand has a 5' end and a 3' end, and wherein the RNA duplex is attached to an adjacent phosphate group at the 5' end of its second strand.

[0449] In some embodiments, equation (XII) is equation (XIIb):

[0450]

[0451] Formula (XIIb)

[0452] And the compound of formula (XIII) is of formula (XIIIa):

[0453]

[0454] Equation (XIIIa)

[0455] The oligonucleotide comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, wherein each of the first strand and the second strand has a 5' end and a 3' end, and wherein the RNA duplex is attached to an adjacent phosphate group at the 5' end of its second strand.

[0456] In some embodiments, the compound of formula (XII) is of formula (XIIc):

[0457]

[0458] Formula (XIIc)

[0459] And the compound of formula (XIII) is of formula (XIIIa):

[0460]

[0461] Equation (XIIIa)

[0462] The oligonucleotide comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, wherein each of the first strand and the second strand has a 5' end and a 3' end, and wherein the RNA duplex is attached to an adjacent phosphate group at the 3' end of its second strand.

[0463] In some embodiments, the compound of formula (XII) is of formula (XIId):

[0464]

[0465] Formula (XIId)

[0466] And the compound of formula (XIII) is of formula (XIIIa):

[0467]

[0468] Equation (XIIIa)

[0469] The oligonucleotide comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, wherein each of the first strand and the second strand has a 5' end and a 3' end, and wherein the RNA duplex is attached to an adjacent phosphate group at the 3' end of its second strand.

[0470] In some embodiments, the compound of formula (XIIIa) is of formula (XIIIb):

[0471]

[0472] Equation (XIIIb)

[0473] In some embodiments, the compound of formula (XII) is prepared by reacting the compound of formula (XIV) and the compound of formula (XV):

[0474]

[0475] Formula (XIV)

[0476]

[0477] Formula (XV)

[0478] R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl;

[0479] R2 is selected from the group consisting of: hydrogen, hydroxyl group, -OC.1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogenated, and nitro groups;

[0480] X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur;

[0481] q, r, s, t, v are independent integers from 0 to 4, provided that:

[0482] (i) q and r cannot both be 0 at the same time; and

[0483] (ii) s, t, and v cannot all be 0 at the same time;

[0484] Z stands for oligonucleotide.

[0485] In some embodiments, the compound of formula (XIV) is of formula (XIVa) or formula (XIVb):

[0486]

[0487] Formula (XIVa)

[0488]

[0489] Formula (XIVb)

[0490] And the compound of formula (XV) is of formula (XVa) or formula (XIVb):

[0491]

[0492] Equation (XVa)

[0493]

[0494] Formula (XVb)

[0495] The oligonucleotide comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first and second strands has a 5' end and a 3' end, and wherein (i) in formula (XVa), the RNA duplex is attached to an adjacent phosphate at the 5' end of its second strand, or (ii) in formula (XVb), the RNA duplex is attached to an adjacent phosphate at the 3' end of its second strand.

[0496] 7. Uses

[0497] This invention relates to the use of compounds and compositions as described anywhere herein.

[0498] The present invention also relates to the use of the compounds described anywhere herein for the preparation of another compound described anywhere herein.

[0499] The present invention further relates to the use of the compositions described anywhere herein for the preparation of another composition described anywhere herein.

[0500] In some embodiments, the use is for the preparation of compounds or compositions as described anywhere herein, wherein R2 = F.

[0501] In some embodiments, the use is for preparing compounds or compositions as described anywhere herein, comprising alternative chain moieties as described anywhere herein. In some embodiments, the use is for preparing compounds or compositions as described anywhere herein, wherein R2 = OH.

[0502] This invention relates to the use of compounds of formula (XII) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIII) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIV) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XV) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0503] This invention relates to the use of compounds of formula (XIIa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIIb) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIIc) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIId) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIIIa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIIIb) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIVa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XIVb) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XVa) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein. This invention relates to the use of compounds of formula (XVb) as described anywhere herein for the preparation of compounds and / or compositions as described anywhere herein.

[0504] This invention relates to compounds or compositions that are obtained or available by methods as described anywhere herein.

[0505] This invention relates to the therapeutic use of compounds and compositions as described anywhere herein.

[0506] Therefore, this invention relates to a pharmaceutical composition comprising a compound as described anywhere herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0507] The present invention also relates to compounds for therapeutic purposes as described anywhere herein. The present invention also relates to compositions for therapeutic purposes as described anywhere herein.

[0508] This invention also covers suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations.

[0509] The compounds of the present invention can be used as research reagents, for example, for diagnosis, treatment and prevention.

[0510] In treatment, the compounds of the present invention can be used to specifically regulate the synthesis of target proteins in cells. This can be achieved by degrading, silencing, or inhibiting the mRNA of the target protein, thereby preventing the formation of the protein. Alternatively, the compounds of the present invention can be used to regulate non-coding DNA or RNA molecules, exerting a regulatory effect on intracellular mechanisms in cells and experimental animals, thereby facilitating functional analysis of the target or assessment of its usefulness as a target for therapeutic intervention.

[0511] In a preferred embodiment, the target protein is in target cells (such as liver cells, particularly hepatocytes) whose surface contains desialyl glycoprotein receptors (ASPGR).

[0512] Therefore, the compounds of the present invention can be used as a treatment for animals or humans suspected of having a disease or disorder that can be alleviated or treated by regulating the DNA or RNA encoding mammalian target polypeptides in the animal or human.

[0513] In a preferred embodiment, the target nucleic acid is a gene, messenger RNA (mRNA), or microRNA (miRNA).

[0514] Furthermore, a method is provided for treating mammals (such as therapeutic humans) suspected of having or susceptible to disease or ailment by administering a therapeutic or preventative amount of one or more compounds or compositions of the present invention.

[0515] The present invention also provides the use of the compounds or conjugates of the present invention as described herein in the manufacture of medicaments for treating disorders or in methods for treating disorders affected by regulatory target nucleic acids.

[0516] The present invention also provides a method for treating a disorder, the method comprising administering to a patient in need a compound according to the invention and / or a pharmaceutical composition according to the invention.

[0517] Examples of untreatable barriers include liver diseases such as hepatitis (including viral hepatitis such as HBV or HCV), hepatic steatosis, atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia such as apolipoprotein B acquired mutation, HDL / LDL cholesterol imbalance, dyslipidemia such as familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD) and coronary heart disease (CHD), cirrhosis, and cancer.

[0518] 8. Definition

[0519] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or expressions used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, for clarity and / or convenience of reference, terms with commonly understood meanings are defined herein, and these definitions contained herein are not necessarily to be construed as indicating a significant difference from the commonly understood meaning in the art compared to definitions of terms commonly understood in the prior art.

[0520] It should be understood that the present invention is not limited to specific compositions or biological systems, which can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used in this specification and the appended claims include plural references.

[0521] As used herein, the term "about" refers to a common range of error for the corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments relating to that value or parameter itself.

[0522] It should be understood that the aspects and embodiments of the invention described herein include those referred to as “comprising,” “consisting of,” and “substantially consisting of.”

[0523] As used herein, the term “and / or” means any one of the items associated with the term, any combination of the items, or all of the items. For example, the phrase “A, B, and / or C” is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A and B or C; B and A or C; C and A or B; A (alone); B (alone); and C (alone).

[0524] 9. Example

[0525] The invention will be more fully understood by referring to the following examples. However, they should not be construed as limiting the scope of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes thereof will be suggested to those skilled in the art and will be included within the spirit and limits of this application and within the scope of the appended claims.

[0526] The following builder is used in the example:

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533] 9.1 Example 1

[0534] -Summary of the Invention

[0535] GalNAc-siRNAs targeting hsHAO1, hsC5, or hsTTR mRNA were synthesized and QC-controlled. The entire siRNA set (excluding the siRNA targeting HAO1) ​​was first studied in HepG2 cells under a dose-response setting using RNAiMAX transfection, and then in primary human hepatocytes under a naked, free-uptake setting in a dose-response analysis.

[0536] Incubating primary human hepatocytes directly with GalNAc-siRNA targeting hsHAO1, hsC5, or hsTTR mRNA resulted in dose-dependent silencing of the target mRNA to varying degrees.

[0537] -Research Objectives

[0538] The purpose of this experimental set is to analyze the in vitro activity of different GalNAc ligands in the context of siRNA targeting three different targets (i.e., hsHAO1, hsC5, or hsTTR mRNA).

[0539] This research package includes (i) determining the development of a set of target-specific bDNA probes designed, synthesized, and tested for each individual target; (ii) identifying cell lines suitable for subsequent screening experiments; (iii) dose-response analysis of all possible siRNAs (via transfection) in one or more human cancer cell lines; and (iv) dose-response analysis of siRNAs in primary human hepatocytes under naked, free-intake conditions. In both conditions, IC50 values ​​and maximum inhibition values ​​should be calculated, and the siRNA study set should then be ranked according to their potency.

[0540] -Materials and methods

[0541] Oligonucleotide synthesis

[0542] Standard solid-phase synthesis methods were used for the chemical synthesis of target siRNA (see Table 1) and controls (see Table 2).

[0543] Cell culture and in vitro transfection experiments

[0544] Cell culture, transfection, and QuantiGene 2.0 branched DNA assays are described below, and siRNA sequences are listed in Tables 1 and 2. HepG2 cells were provided by the American Center for Tissue Culture Collection (ATCC) (HB-8065, lot number: 63176294) and cultured in Eagle Minimal Basal Medium supplemented with 10% Fetal Bovine Serum (FCS) prepared by ATCC. Primary human hepatocytes (PHH) were derived from Primacyt (Schwerin, Germany) (lot number: CyHuf19009HEc). Cells were obtained from malignant glioblastoma tumors via explant technology. All cells used in this study were cultured at 37°C in a humidified incubator with an atmosphere containing 5% CO2.

[0545] To transfect HepG2 cells with siRNAs targeting hsC5 or hsTTR (and controls), cells were seeded at a density of 20,000 cells / well in standard 96-well tissue culture plates. Cells were transfected with siRNA using the commercially available transfection reagent RNAiMAX (Invitrogen / Life Technologies) according to the manufacturer's instructions. A 10-point dose-response assay was performed in HepG2 cells using 20 candidates (11x hsC5, 9x hsTTR) at final siRNA concentrations of 24 nM, 6 nM, 1.5 nM, 0.4 nM, 0.1 nM, 0.03 nM, 0.008 nM, 0.002 nM, 0.0005 nM, and 0.0001 nM.

[0546] Dose-response analysis in PHH was performed by directly incubating cells in a naked, free-up environment, starting with a maximum final concentration of 1.5 µM siRNA, followed by 500 nM, and then decreasing sequentially in two-fold dilution steps.

[0547] Control wells were transfected into HepG2 cells or directly incubated with primary human hepatocytes at the highest test siRNA concentration studied on the corresponding plates. Table 2 summarizes and lists all control siRNAs included in different project phases following cell-mimicking treatment. For each siRNA and control, at least four wells were transfected / directly incubated in parallel, and individual data points were collected from each well.

[0548] After transfection and incubation with siRNA for 24 hours, the culture medium was removed and HepG2 cells were lysed in a lysis mixture (1 volume of lysis buffer plus 2 volumes of nuclease-free water), followed by incubation at 53°C for at least 45 minutes. In the case of PHH, the plating medium was removed 5 hours after cell treatment, and 50 µl of complete maintenance medium was added to each well. The medium was changed every 24 hours in this manner until the total incubation period of 72 hours. At the 4-hour or 72-hour time point, the cell culture supernatant was removed, and 200 µl of lysis mixture supplemented with 1:1000 v / v proteinase K was added.

[0549] Branched DNA (bDNA) assays were performed according to the manufacturer's instructions. After incubation in the dark for 30 minutes in the presence of substrate, luminescence was read using a 1420 luminescent counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jügesheim, Germany). For each well, the target mRNA level was normalized to hsGAPDH mRNA level. The activity of any siRNA is expressed as a percentage of the target mRNA concentration (normalized to hsGAPDH mRNA) in the treated cells relative to the mean target mRNA concentration (normalized to hsGAPDH mRNA) in the control wells.

[0550] Measurement and Development

[0551] The QuantiGene 2.0 branched DNA (bDNA) probe set is specifically designed and synthesized for Homo sapiens GAPDH, AHSA1, hsHAO1, hsC5, and hsTTR. The bDNA probe set was initially tested via bDNA analysis according to the manufacturer's instructions, evaluating the levels of target mRNAs in the following human and monkey cancer cell lines adjacent to primary human hepatocytes at two different lysate amounts (i.e., 10 µl and 50 µl): SJSA-1, TF1, NCI-H1650, Y-79, Kasumi-1, EAhy926, Caki-1, Colo205, RPTEC, A253, HeLaS3, Hep3B, BxPC3, DU145, THP-1, NCI-H460, IGR37, LS174T, Be(2)-C, SW 1573, NCI-H358, TC71, 22Rv1, BT474, HeLa, KBwt, Panc-1, U87MG, A172, C42, HepG2, LNCaP, PC3, SupT11, A549, HCT116, HuH7, MCF7, SH-SY5Y, HUVEC, C33A, HEK293, HT29, MOLM 13, and SK-MEL-2. Wells containing only bDNA probe sets without added cell lysates were used to monitor technical background and noise signals.

[0552] -result

[0553] Identifying cell types suitable for screening GalNAc-siRNA

[0554] Figures 1 to 3 This study presents mRNA expression data for three targets (hsC5, hsHAO1, and hsTTR) in lysates from a diverse set of human cancer cell lines plus primary human hepatocytes. The cell number / lysate volume was identical for each cell line tested, which is necessary to allow for comparisons of expression levels between different cell types. Figure 1 The hsC5 mRNA expression data for all cell types tested are shown.

[0555] We also screened for the same cell type expressing hsHAO1 mRNA, and the results showed that... Figure 2 A portion of the bar chart.

[0556] Finally, a suitable cell type was identified that will allow for screening of GalNAc-siRNAs targeting hsTTR, and the corresponding data are... Figure 3 Part of it.

[0557] In summary, the expression levels of all three target mRNAs were sufficiently high in primary human hepatocytes (PHH). Furthermore, HepG2 cells could be used to screen for GalNAc-siRNAs targeting hsC5 and hsTTR mRNAs, whereas cancer cell lines suitable for testing siRNAs specific to hsHAO1 mRNA could not be identified.

[0558] Dose-response analysis of GalNAc-siRNA targeting hsTTR in HepG2 cells

[0559] Following transfection optimization, HepG2 cells were transfected with the entire GalNAc-siRNA set targeting hsTTR (see Table 1) using RNAiMAX in a dose-response setting. The highest siRNA assay final concentration was 24 nM, decreased in nine 4-fold dilution steps. Experiments were completed at 4 and 24 hours post-transfection of HepG2 cells. Table 3 lists the activity data of all GalNAc-siRNAs targeting hsTTR from all studies.

[0560] Table 3: Targets, incubation times, external IDs, IC20 / IC50 / IC80 values, and maximum inhibition of siRNAs targeting hsTTR in HepG2 cells. The list is sorted by external ID, with 4-hour incubation at the top and 24-hour incubation at the bottom.

[0561]

[0562] The results of 24-hour incubation also showed Figure 4A -D

[0563] Typically, transfection of HepG2 cells with hsTTR-targeting siRNA results in silencing of the target mRNA, generally spanning the entire activity range from 0% silencing to maximal inhibition. Data generated at 24 hours post-transfection are more robust and have lower standard deviations compared to data generated only at 4 hours post-transfection. Furthermore, the degree of target knockdown typically increases over time (from 4 hours to 24 hours of incubation). hsTTR GalNAc-siRNAs that achieve >95% silencing of the target mRNA have been identified, with IC50 values ​​in the low double-digit pM range.

[0564] Dose-response analysis of GalNAc-siRNA targeting hsC5 in HepG2 cells

[0565] The second target, hsC5 mRNA, was tested using RNAiMAX transfection of HepG2 cells at the same dose-response settings (in any case, minimal difference in the final concentration of the siRNA assay). GalNAc-siRNA and hsTTR siRNA had the same residence / position / GalNAc-ligand variations, but the sequence pair was specific to the target hsC5 mRNA. Additionally, the assay set of the studied molecules included two additional variants of siRNA XD-30951. One variant was a fraction of the full-length product (FLP) minus 2 Da, and the other was a fractionated pure FLP.

[0566] Table 4: Targets, incubation times, external IDs, IC20 / IC50 / IC80 values, and maximum inhibition of siRNAs targeting hsC5 in HepG2 cells. The list is sorted by external ID, with 4-hour incubation at the top and 24-hour incubation at the bottom.

[0567]

[0568] The results of 24-hour incubation also showed Figure 5A -D

[0569] Following transfection of HepG2 cells with a set of GalNAc-siRNAs specific to hsC5, dose-dependent silencing of the target hsC5 mRNA was observed. Some knockdown was detectable as early as 4 hours post-transfection, with even higher levels of silencing observed after a longer incubation period (i.e., 24 hours). The GalNAc-siRNA that silenced nearly 90% of the target mRNA was identified, with IC50 values ​​in the low single-digit pM range.

[0570] Identifying primary human hepatocyte batches suitable for testing all GalNAc-siRNAs

[0571] Dose-response analysis of two GalNAc-siRNA sets in the human cancer cell line HepG2 should demonstrate (and ensure) that all novel GalNAc- / linker / position / cap variants are indeed substrates that efficiently bind to AGO2 and are loaded into RISC, and additionally capable of functioning in RNAi-mediated target mRNA cleavage. However, to test whether targeting GalNAc ligand derivatives allows for efficient uptake into hepatocytes, dose-response analysis experiments should be performed in primary human hepatocytes using a naked, free-uptake setting. Hepatocytes do express desialylate glycoprotein receptor (ASGR1) at high levels, and the liver typically uses this receptor to remove target glycoproteins from circulation. It is now known that certain types of GalNAc ligand-conjugated oligonucleotides (e.g., siRNA or ASO) are recognized by this high-turnover receptor and efficiently taken up into the cytoplasm via clathrin-coated vesicles and transported to the endosome compartment. Endosomal escape is considered the rate-limiting step in oligonucleotide delivery.

[0572] Intermediate assays were performed to develop a assay that tested the expression levels of relevant target genes (hsC5, hsTTR, hsHAO1, hsGAPDH, and hsAHSA1) in different batches of primary human hepatocytes. Primacyt (Schwerin, Germany) provided three different batches of primary human hepatocytes for testing: BHuf16087, CHF2101, and CyHuf19009. Cells were seeded in collagen-coated 96-well tissue culture plates and incubated for 0, 24, 48, and 72 hours, followed by cell lysis and bDNA analysis to monitor target mRNA levels. Figure 6 The absolute mRNA expression data of all three targets (hsTTR, hsC5, and hsHAO1) ​​in primary human hepatocyte batches BHuf16087, CHF2101, and CyHuf19009 are shown. The mRNA expression levels of hsGAPDH and hsAHSA1 are shown below. Figure 7 As shown.

[0573] exist Figure 6 and Figure 7 In the dataset, the left column of each triplet is BHuf16087, the middle column is CHF2101, and the right column is CyHuf19009.

[0574] Overall, the mRNA expression of all three targets in primary human hepatocyte batches BHuf16087 and CyHuf19009 was sufficiently high after 72 hours to proceed with bDNA assays. Due to the limited available vials for further experiments, we continued with batch CyHuf19009.

[0575] Dose-response analysis of GalNAc-siRNA targeting hsHAO1 in PHH

[0576] After identifying a suitable batch of primary human hepatocytes (PHH) (CyHuf19009), a naked free uptake assay was performed on the GalNAc-siRNA targeting hsHAO1 listed in Table 1. The highest siRNA final concentration tested was 1.5 µM, followed by 500 nM, decreasing in eight two-fold serial dilution steps to the lowest siRNA final concentration of 1.95 nM. The experiment ended 4 hours and 72 hours after direct incubation of PHH cells. Table 5 lists the activity data of all the GalNAc-siRNAs targeting hsHAO1 studied. Table 2 summarizes and lists all the control siRNAs included in this experiment.

[0577] Table 5: Targets, incubation times, external IDs, IC20 / IC50 / IC80 values, and maximum inhibition of GalNAc-siRNAs targeting hsHAO1 in primary human hepatocytes (PHH). The list is organized by external ID, with 4-hour incubation and 72-hour incubation listed in the upper and lower parts respectively.

[0578]

[0579] The results of the 72-hour incubation are also shown in Figure 8A -D.

[0580] Naked free uptake of GalNAc-siRNA targeting hsHAO1 did not result in significant on-target silencing within 4 hours, but on-target silencing was visible within the range of 35.5% to 58.1% maximum inhibition after 72-hour incubation.

[0581] Dose-response analysis of GalNAc-siRNA targeting hsC5 in PHH

[0582] The second target, hsC5 mRNA, was tested in PHH by naked free uptake under the same dose-response setting. The GalNAc-siRNA had the same linker / position / GalNAc-ligand variation as hsTTR and hsHAO1 tested previously in the assay, except for the sequence specific to on-target hsC5 mRNA. The sequences of the GalNAc-siRNAs targeting hsC5 and all the sequences and information regarding the control siRNAs are listed in Table 1 and Table 2 respectively. The experiment ended 4 hours and 72 hours after direct incubation of PHH. Table 6 lists the activity data of all the GalNAc-siRNAs targeting hsC5 studied.

[0583] Table 6: Targets, incubation times, external IDs, IC20 / IC50 / IC80 values, and maximum inhibition of GalNAc-siRNAs targeting hsC5 in PHH. The list is organized by external ID, with 4-hour incubations and 72-hour incubations listed in the upper and lower parts, respectively.

[0584]

[0585] Results for the 72-hour incubation are also shown in Figure 9A -D.

[0586] No significant on-target silencing of GalNAc-siRNA was seen after 4 hours of incubation. Data generated after a 72-hour incubation period showed more robust on-target silencing with up to 65.5% maximum inhibition.

[0587] Dose-response analysis of GalNAc-siRNAs targeting hsTTR in PHH

[0588] The last target, hsTTR mRNA, was again tested in PHH with the same dose-response setup as for the targets hsHAO1 and hsC5 with naked free uptake, with the only difference being the use of a specific siRNA sequence for on-target hsTTR mRNA (see Table 1).

[0589] The experiment was terminated after 72 hours of direct incubation in PHH. Table 7 lists the activity data for all GalNAc-siRNAs targeting hsTTR that were studied.

[0590] Table 7: Targets, incubation times, external IDs, IC20 / IC50 / IC80 values, and maximum inhibition of GalNAc-siRNAs targeting hsTTR in primary human hepatocytes (PHH). The list is organized by external ID.

[0591]

[0592] Results are also shown in Figure 10A -D.

[0593] Naked free uptake of GalNAc-siRNAs targeting hsTTR did result in significant on-target silencing within 72 hours, ranging from 46% to 82.5% maximum inhibition. The hsTTR GalNAc-siRNAs were identified to silence the on-target mRNA with IC50 values in the low double-digit nM range.

[0594] - Summary and Discussion

[0595] The scope of this study is to analyze the in vitro activity of the GalNAc ligand according to the invention in the context of siRNAs targeting three different on-target mRNAs (i.e., hsHAO1, hsC5, and hsTTR). The target-specific siRNA set consists of siRNAs (each with two different antisense strands) with different linker / cap / modification / GalNAc ligand chemistry.

[0596] For all targets, the GalNAc-siRNAs in Table 1 were identified as exhibiting high overall potency and low IC50 values.

[0597] 9.2 Example 2

[0598] Synthetic route

[0599] i) Synthesis of the fused structural unit TriGalNAc

[0600] Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. Compounds were observed under UV light (254 nm) or after spraying with 5% H₂SO₄ in methanol (MeOH) or according to Stahl's ninhydrin reagent (from Sigma-Aldrich), followed by heating. A Biotage Isolera equipped with a dual variable UV wavelength detector (200 nm–400 nm) was used. The One Rapid Chromatography instrument uses Biotage Sfär silica columns of 10 g, 25 g, 50 g, or 100 g (Uppsala, Sweden) for rapid chromatography.

[0601] All moisture-sensitive reactions were performed under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK Scientific.

[0602] HPLC / ESI-MS was performed on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQPlus mass spectrometer using a Waters Acquity UPLC Protein BEH C4 column (300 Å, 1.7 µm, 2.1 x 100 mm) at 60 °C. The solvent system consisted of solvent A (H₂O containing 0.1% formic acid) and solvent B (acetonitrile (ACN) containing 0.1% formic acid). A gradient of 5%–100% B was used over 15 minutes at a flow rate of 0.4 mL / min. Detector and conditions: Corona overcharged electrosol detection (from ESA). Nebulizer temperature: 25 °C. N₂ pressure: 35.1 psi. Filter: Corona.

[0603] 1 H and 13 C NMR spectra at room temperature on a Varian spectrometer at 500 MHz ( 1 H NMR) and 125 MHz ( 13 Recorded under CNMR. Chemical shifts are given in ppm, with reference to solvent residue peaks (CDCl3 – 1 H NMR: δ at 7.26 ppm and 13 CNMR δ at 77.2 ppm; DMSO-d6 – 1 H NMR: δ at 2.50 ppm and 13 C NMR δ at 39.5 ppm. Coupling constants are given in Hertz. Signal splitting modes are described as singlet (s), doublet (d), triplet (t), or multiplet (m).

[0604] ii) Synthetic route of the fused structural unit TriGalNAc

[0605]

[0606] Preparation of Compound 2: D-galactosamine pentaacetate (3.00 g, 7.71 mmol, 1.0 eq.) was dissolved in anhydrous dichloromethane (DCM) (30 mL) under argon atmosphere, and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.28 g, 19.27 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature for 3 hours. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated NaHCO3 aqueous solution (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the title compound as a yellow oil, which was purified by rapid chromatography (gradient elution: 0–10% MeOH in DCM, 10 CV). The product as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM)) was obtained.

[0607]

[0608] Preparation of Compound 4: Compound 2 (2.30 g, 6.98 mmol, 1.0 eq.) and azide-PEG3-OH (1.83 g, 10.5 mmol, 1.5 eq.) were dissolved in anhydrous DCM (40 mL) under argon atmosphere, and molecular sieve 3Å (5 g) was added to the solution. The mixture was stirred at room temperature for 1 hour. Then, TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was added to the mixture and the reaction was stirred overnight. The molecular sieve was filtered, and the filtrate was diluted with DCM (100 mL) and washed with cold saturated NaHCO3 aqueous solution (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude substance was purified by rapid chromatography (gradient elution: 0-3% MeOH in DCM, 10 CV) to give the title product (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM)) as a pale yellow oil. MS: C 20 H 32 N4O 11 The calculated value is 504.21. The measured value is 505.4. 1H NMR (500 MHz, CDCl3) δ 6.21-6.14 (m, 1H), 5.30 (dd, J = 3.4, 1.1 Hz, 1H), 5.04 (dd, J = 11.2, 3.4 Hz, 1H), 4.76 (d, J = 8.6Hz, 1H), 4.23-4.08 (m, 3H), 3.91-3.80 (m, 3H), 3.74-3.59 (m, 9H), 3.49-3.41(m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.97 (d, J = 4.2 Hz, 6H). 13 C NMR (125MHz, CDCl3) 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).

[0609]

[0610] Preparation of Compound 5: Compound 4 (1.00 g, 1.98 mmol, 1.0 eq.) was dissolved in a mixture of ethyl acetate (EtOAc) and MeOH (30 mL 1:1 v / v), and Pd / C (100 mg) was added. The reaction mixture was degassed using a vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The reaction mixture was filtered through diatomaceous earth and washed with EtOAc (30 mL). The solvent was removed under reduced pressure to give the title compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM)). This compound was ready for use without further purification. MS: C 20 H 34 N2O 11 The calculated value is 478.2. The measured value is 479.4.

[0611]

[0612] Preparation of compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM and water (40 mL, 1:1 v / v), and Na₂CO₃ (0.18 g, 1.7 mmol, 0.25 eq.) was added while stirring vigorously. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 hours. The reaction mixture was diluted with CH₂Cl₂ (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na₂SO₄. The solvent was removed under reduced pressure, and the crude product was purified by rapid chromatography (gradient elution: 0–10% EtOAc in cyclohexane, 12 CV) to give the title compound (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane)) as a pale yellow oil. MS: C 33 H 53 NO 11 The calculated value is 639.3. The measured value is 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 (125MHz, 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).

[0613]

[0614] Preparation of compound 8: Cbz-NH-tri-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL) under argon atmosphere, trifluoroacetic acid (TFA, 1 mL) was added, and the reaction was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was co-evaporated three times with toluene (5 mL) and dried under high vacuum to give the compound (0.183 g, 98%) as its TFA salt. This compound was ready for use without further purification. MS: C 21 H 29 NO 11 The calculated value is 471.6. The measured value is 472.4.

[0615]

[0616] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH2 5 (3.56 g, 7.44 mmol, 5.0 eq.) were dissolved in N,N-dimethylformamide (DMF) (25 mL). Then, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)ureonium hexafluorophosphate (HBTU) (2.78 g, 7.44 mmol, 5.0 eq.), 1-hydroxybenzotriazine hydrate (HOBt) (1.05 g, 7.44 mmol, 5.0 eq.), and N,N-diisopropylethylamine (DIPEA) (2.07 mL, 11.9 mmol, 8.0 eq.) were added to the solution and the reaction was stirred for 72 hours. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and washed with a saturated aqueous solution of NaHCO3 (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude material was purified by rapid chromatography (gradient elution: 0–5% MeOH in DCM, 14 CV). A pale yellow oily product was obtained (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM)). MS: C 81 H 125 N7O 41 The calculated value is 1852.9. The measured value is 1854.7. 1H NMR (500 MHz, DMSO-d6)δ 7.90-7.80 (m, 10H), 7.65-7.62 (m, 4H), 7.47-7.43 (m, 3H), 7.38-7.32 (m,8H), 5.24-5.22 (m, 3H), 5.02-4.97 (m, 4H), 4.60-4.57 (m, 3 H), 4.07-3.90 (m10H), 3.67-3.36 (m, 70H), 3.23-3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H),1.89 (s, 11H), 1.80-1.78 (m, 17H). 13 C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7(CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[0617]

[0618] Preparation of Compound 10: Trianthopteran GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) was dissolved in MeOH (15 mL), and 3 drops of acetic acid (AcOH) and Pd / C (30 mg) were added. The reaction mixture was degassed using a vacuum / argon cycle (3x) and hydrogenated overnight under balloon pressure. After the reaction was complete, mass spectrometry was performed, and the resulting mixture was filtered through a diatomaceous earth thin-layer filter. The solvent was evaporated, and the resulting residue was dried under high vacuum and used in the next step without further purification. A product (0.24 g, quantitative yield) was given as a pale yellow oil. MS: C 73 H 119 N7O 39 The calculated value is 1718.8. The measured value is 1719.3.

[0619]

[0620] Preparation of Compound 11: Commercially available bis(N-hydroxysuccinimide) octanoic acid (3.67 g, 9.9 mmol, 1.0 eq.) was dissolved in DMF (5 mL) and triethylamine (1.2 mL) was added. A solution of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0 eq.) in DMF (5 mL) was added dropwise to this solution. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified by rapid chromatography (gradient elution: 0–5% MeOH in DCM, 16 CV). The product was given as a white solid (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM)). MS: C 15 H 23 The calculated value of N5O5 is 353.4. The measured value is 354.3.

[0621]

[0622] Preparation of TriGalNAc (12): Compound 10 (0.35 g, 0.24 mmol, 1.0 eq.) and compound 11 (0.11 g, 0.31 mmol, 1.5 eq.) of the trianthraquinone GalNAc were dissolved in DCM (5 mL) under argon atmosphere, and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reaction was stirred overnight at room temperature. After removing the solvent under reduced pressure, the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was evaporated and the resulting crude product was purified by rapid chromatography (elution gradient: 0–10% MeOH in DCM, 20 CV) to give the title compound (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM)) as a white, fluffy solid. MS: C 84 H 137 N 11 O 41 The calculated value is 1957.1. The measured value is 1959.6.

[0623] Compound 12 was used in the preparation of subsequent oligonucleotide conjugates using "click chemistry".

[0624] iii) Oligonucleotide synthesis

[0625] Table 8:

[0626]

[0627] Af, Cf, Gf, Uf: 2'-F RNA nucleotides

[0628] a, c, g, u: 2'-O-Me RNA nucleotides

[0629] dT: DNA nucleotides

[0630] s: Phosphoric acid

[0631] invabasic: 1,2-dideoxyribose

[0632] NH2-DEG: Aminoethoxyethyl linker

[0633] NH2C12: Amino-dodecyl linker

[0634] NH2C6: Aminohexyl linker

[0635] Oligonucleotides were synthesized on the solid phase using the phosphoramide method. Depending on the scale, either Mermade 12 (BioAutomation Corporation) or ÄKTA Oligopilot (GE Healthcare) was used.

[0636] Synthesis was carried out on a commercially available solid support made of controllable porous glass loaded with invabasic (CPG, 480 Å, 86 μmol / g; LGC Biosearch catalog BCG-1047-B) or 2'-FA (CPG, 520 Å, 90 μmol / g; LGC Biosearch catalog BCG-1039-B) or NH2C6 (CPG, 520 Å, 85 μmol / g; LGC Biosearch catalog BCG-1397-B) or GalNAc (CPG, 500 Å, 57 μmol / g; Primetech) or 2'-O-methyl C (CPG, 500 Å, 84 μmol / g; LGC Biosearch catalog BCG-10-B) or 2'-O-methyl A (CPG, 497 Å, 85 μmol / g; LGC Biosearch catalog BCG-10-B) or LGC Biosearch catalog BCG-10-B. Biosearch catalog number BCG-1029-B) or dT (CPG, 497 Å, loading of 87 μmol / g; LGC Biosearch catalog number BCG-1055-B).

[0637] 2'-O-Me, 2'-F RNA phosphorous amide, and auxiliary reagents were purchased from SAFC Proligo (Hamburg, Germany).

[0638] Specifically, the following 2'-O-methylphosphoramides were used: 5'-(4,4'-dimethoxytriphenylmethyl)-N-benzoyl-adenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-benzoyl-cytidine 2'-O-methyl-3'-[(2-cyanoethyl)-(N, N-Diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-dimethylformamidin-guanosine, 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-uridine, 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide.

[0639] The following 2'-F phosphoramids were used: 5'-dimethoxytriphenylmethyl-N-benzoyl-deoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-dimethoxytriphenylmethyl-N-acetyl-deoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-dimethoxytriphenylmethyl-N-isobutyryl-deoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid and 5'-dimethoxytriphenylmethyl-deoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid.

[0640] To introduce the desired amino linker at the 5' end of the oligonucleotide, 2-[2-(4-monomethoxytriphenylmethyl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramide (Glen Research catalog number 1905) and 12-(trifluoroacetylamino)dodecyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide (ChemGenes catalog number CLP-1575) were used. 5-O-dimethoxytriphenylmethyl-1,2-dideoxyribose-3-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide (ChemGenes catalog number ANP-1422) was also used.

[0641] All structural units were dissolved in anhydrous acetonitrile (100 mM (Mermade12) or 200 mM (ÄKTA Oligopilot)) containing a molecular sieve (3 Å), except for 2'-O-methyluridine phosphoramidite, which was dissolved in 50% anhydrous DCM in anhydrous acetonitrile. Iodine (50 mM, in pyridine / H2O, 9:1 v / v) was used as the oxidizing agent. 5-Ethiotetrazole (ETT, 500 mM, in acetonitrile) was used as the activating agent solution. Thiolation to introduce thiophosphate linkages was carried out using 100 mM hydroxanthin (TCI catalog number 6846-35-1, in acetonitrile / pyridine, 4:6 v / v).

[0642] Unless otherwise specified, the coupling time was 5.4 minutes. The 5' amino group was incorporated into the sequence using a double coupling step, with each coupling session lasting 11 minutes (total coupling time 22 minutes). The oxidant contact time was set to 1.2 minutes, and the thiolation time was 5.2 minutes.

[0643] The sequence was synthesized by removing the final DMT group, except for the MMT group in the NH2DEG sequence.

[0644] At the end of the synthesis, oligonucleotides were cleaved from the solid support at 6°C for 16 hours using a 1:1 volume mixture of 28%–30% ammonium hydroxide solution (Sigma-Aldrich, catalog number 221228) and 40% methylamine aqueous solution (Sigma-Aldrich, catalog number 8220911000). The solid support was then filtered off, the filter was thoroughly washed with H₂O, and the volume of the combined solutions was reduced by evaporation under reduced pressure. The pH of the resulting solution was adjusted to pH 7 with 10% AcOH (Sigma-Aldrich, catalog number A6283).

[0645] The crude material was purified by reversed-phase (RP) HPLC or anion-exchange (AEX) HPLC.

[0646] RP HPLC purification was performed on an ÄKTA Pure instrument (GE Healthcare) using an XBridge C18 Prep 19 x 50 mm column (Waters). Buffer A was 100 mM triethylammonium acetate (TEAAc, Biosolve) 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. UV traces were recorded at 280 nm. A gradient from 0% B to 100% B was used over 120 column volumes. Appropriate fractions were combined and precipitated in a refrigerator using 3 M sodium acetate (NaOAc) (Sigma-Aldrich) pH 5.2 and 85% ethanol (VWR). The precipitate was separated by centrifugation, redissolved in water (50 mL), treated with 5 M NaCl (5 mL), and desalted by size exclusion HPLC on an Äkta Pure instrument using a 50 x 165 mm ECO column (YMC, Dinslaken, Germany) packed with Sephadex G25-Fine resin (GE Healthcare).

[0647] AEX HPLC purification was performed on an ÄKTA Pure instrument (GE Healthcare) using a TSK gel SuperQ-5PW 20x 200 mm (BISCHOFF Chromatography) lens. Buffer A was 20 mM sodium phosphate (Sigma-Aldrich) pH 7.8, and buffer B was the same as buffer A but with the addition of 1.4 M sodium bromide (Sigma-Aldrich). A flow rate of 10 mL / min and a temperature of 60 °C were used. UV traces were recorded at 280 nm. A gradient of 10% B to 100% B was used over 27 column volumes. Appropriate fractions were combined and precipitated in a refrigerator with 3 M NaOAc, pH 5.2, and 85% ethanol. The precipitate was separated by centrifugation, redissolved in water (50 mL), treated with 5 M NaCl (5 mL), and desalted by size exclusion chromatography.

[0648] The MMT groups were removed using a 25% aqueous acetic acid solution. Once the reaction was complete, the solution was neutralized, and the sample was desalted by size exclusion chromatography.

[0649] Single-stranded molecules were analyzed by analytical LC-MS on a 2.1 x 50 mm XBridge C18 column (Waters) using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with either an LCQ Deca XP-plus Q-ESI-TOF mass spectrometer (Thermo Finnigan) or a CompactESI-Qq-TOF mass spectrometer (Bruker Daltonics). Buffer A consisted of 16.3 mM triethylamine, 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and 1% MeOH in H₂O, while buffer B contained buffer A in 95% MeOH. A flow rate of 250 µL / min and a temperature of 60 °C were used. UV traces were recorded at 260 nm and 280 nm. A gradient of 1%–40% B was used over 0.5 min, followed by a gradient of 40%–100% B over 13 min. Methanol (LC-MS grade), water (LC-MS grade), 1,1,1,3,3,3-hexafluoro-2-propanol (ultra-pure grade) and triethylamine (ultra-pure grade) were purchased from Sigma-Aldrich.

[0650]

[0651] General conditions for MFCO conjugation: The amine-modified single chain was dissolved at 700 OD / mL in 50 mM carbonate / bicarbonate buffer pH 9.6 / dimethyl sulfoxide (DMSO) 4:6 (v / v), and a one-molar equivalent of 35 mM MFCO-C6-NHS ester (Berry & Associates, catalog number LK 4300) in DMF was added to this solution. The reaction was carried out at room temperature, and after 1 hour, another one-molar equivalent of MFCO solution was added. The reaction was allowed to proceed for another hour and monitored by LC / MS. A minimum two-molar equivalent excess of MFCO NHS ester reagent relative to the amine-modified oligonucleotide is required to achieve quantitative consumption of the starting material. The reaction mixture was diluted 15-fold with water, filtered through a 1.2 µm filter from Sartorius, and then purified by reverse-phase HPLC on an Äkta Pure instrument (GE Healthcare).

[0652] Purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM TEEAAc 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. UV traces were recorded at 280 nm. A 0-100% B gradient was applied over 60 column volumes.

[0653] Fractions containing full-length conjugated oligonucleotides were combined and precipitated in a refrigerator using 3 M NaOAc, pH 5.2, and 85% ethanol. The collected precipitate was then dissolved in water. The samples were desalted by size exclusion chromatography and concentrated using a Speed-Vac concentrator to obtain the conjugated oligonucleotides with a separation yield of 40%–80%.

[0654] Table 9:

[0655]

[0656] v) TriGalNAc (GalNAc-T1) ligation at the 5' or 3' end

[0657] 5'-GalNAc-T1 conjugate

[0658]

[0659] 3'-GalNAc-T1 conjugate

[0660]

[0661] The general procedure for TriGalNAc conjugation was as follows: The MFCO-modified single chain was dissolved in water at 2000 OD / mL, and an equivalent of compound 12 (10 mM) in DMF solution was added to this solution. The reaction was carried out at room temperature, and after 3 hours, 0.7 molar equivalents of compound 12 solution were added. The reaction was allowed to proceed overnight and the completion was monitored by LCMS. The conjugate was diluted 15-fold in water, filtered through a 1.2 µm filter from Sartorius, and then purified by RP HPLC on an Äkta Pure instrument (GE Healthcare).

[0662] RP HPLC purification was performed using an XBridge C18 Prep 19 x 50 mm column from Waters. Buffer A was 100 mM triethylammonium acetate at 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. UV traces were recorded at 280 nm. A 0-100% B gradient was employed over 60 column volumes.

[0663] Fractions containing full-length conjugated oligonucleotides were combined and precipitated in a refrigerator using 3 M NaOAc, pH 5.2, and 85% ethanol. The collected precipitate was dissolved in water to obtain an oligonucleotide solution of approximately 1000 OD / mL. O-acetate was removed by adding 20% ​​ammonia. The quantitative removal of these protecting groups was verified by LC-MS.

[0664] The conjugates were desalted using size exclusion chromatography on an Äkta Pure (GE Healthcare) instrument with Sephadex G25 Fine resin (GE Healthcare) to obtain conjugated nucleotides, with a separation yield of 50%-70%.

[0665] Table 10:

[0666]

[0667] vi) Double-chain annealing

[0668] To generate the desired siRNA duplex, the two complementary strands were annealed by combining equimolar aqueous solutions of the two strands. The mixture was placed in a water bath at 70°C for 5 minutes, followed by cooling to ambient temperature over 2 hours. The duplex was then lyophilized for 2 days and stored at -20°C.

[0669] The doublet was analyzed on a Superdex™ 75 Increase 5 / 150 GL column (5 x 153–158 mm, Cytiva) using analytical SEC HPLC on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system. The mobile phase consisted of 1x PBS containing 10% acetonitrile. An isocratic gradient was run over 10 min at a flow rate of 1.5 mL / min at room temperature. UV traces were recorded at 260 nm and 280 nm. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[0670] The prepared GalNAc conjugates are summarized in the table below. These target three different genes. siRNA encoding, as well as corresponding single-stranded and double-stranded sequence information, and purity were collected.

[0671] Table 11:

[0672]

[0673]

[0674] The following scheme further elaborates on the synthetic route:

[0675] Option 1:

[0676]

[0677] Option 2:

[0678]

[0679] Option 3:

[0680]

[0681] Option 4:

[0682]

[0683] Option 5:

[0684]

[0685] Example 3

[0686] Mouse data from GalNAc-siRNA constructs ETX005 and ETX014

[0687] ETX005 (targeting HAO1 mRNA) T1a reverse abase-free

[0688] An in vivo mouse pharmacology study was conducted, which showed that a single subcutaneous dose of up to 3 mg / kg of GalNAc-conjugated siRNA ETX005 resulted in knockdown of HAO1 mRNA in liver tissue and a corresponding increase in serum glycolic acid levels.

[0689] Male C57BL / 6 mice, approximately 8 weeks old, were randomly assigned to groups of 21 mice each. On day 0 of the study, animals received a single subcutaneous dose of 0.3 mg / kg or 3 mg / kg GalNAc-siRNA dissolved in saline (sterile 0.9% sodium chloride), or saline alone as a control. On days 1, 2, 4, 7, 14, 21, and 28 of the study, 3 mice from each group were euthanized, and serum and liver samples were collected.

[0690] Serum was extracted from a group of 5 untreated mice on day 0 to provide baseline measurements of glycolic acid concentration.

[0691] Serum was stored at -80°C until further analysis. Liver samples (approximately 50 mg) were treated with RNAlater and stored overnight at 4°C, then stored at -80°C.

[0692] Quantitative real-time PCR was used to analyze HAO1 mRNA (Thermo assay ID Mm00439249_m1) and housekeeping gene GAPDH mRNA (Thermo assay ID Mm99999915_g1) in liver samples. The ΔΔCt method was used to calculate the change in HAO1 expression normalized to GAPDH relative to the saline control group.

[0693] Seven days later, a single dose of 3 mg / kg ETX005 inhibited HAO1 mRNA expression by more than 80%. Figure 11 The inhibition of HAO1 expression was durable, with a single 3 mg / kg dose of ETX005 maintaining greater than 60% inhibition of HAO1 mRNA at the end of the study on day 28. A single dose of 0.3 mg / kg ETX005 also inhibited HAO1 expression compared to the saline control group, with HAO1 expression levels only reaching normal levels on day 28 of the study.

[0694] It is expected that inhibition of HAO1 mRNA expression will lead to an increase in serum glycolic acid levels. Serum glycolic acid concentration was measured using LC-MS / MS. Figure 12 A single 3 mg / kg dose of ETX005 resulted in a significant increase in serum glycolic acid concentration, peaking at 14 days post-administration and remaining above baseline (day 0) and the saline control until the end of the study on day 28. A single 0.3 mg / kg dose of ETX005 showed serum glycolic acid concentrations above baseline and those seen in the saline control, with a smaller and more transient increase, indicating that even very small doses can inhibit HAO1 mRNA to an order of magnitude sufficient to affect the concentration of metabolic biomarkers in serum.

[0695] ETX014 (targeting C5 mRNA) T1a reverse baseless

[0696] An in vivo mouse pharmacology study was conducted, which showed that a single subcutaneous dose of up to 3 mg / kg of GalNAc-conjugated siRNA ETX014 resulted in knockdown of C5 mRNA in liver tissue and a decrease in serum C5 protein concentration.

[0697] Male C57BL / 6 mice, approximately 8 weeks old, were randomly assigned to groups of 21 mice each. On day 0 of the study, animals received a single subcutaneous dose of GalNAc-siRNA dissolved in saline (sterile 0.9% sodium chloride) at doses of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, or saline alone as a control. On days 1, 2, 4, 7, 14, 21, and 28 of the study, three mice from each group were euthanized, and serum and liver samples were collected.

[0698] Serum was stored at -80°C until further analysis. Liver samples (approximately 50 mg) were treated with RNAlater and stored overnight at 4°C, then stored at -80°C.

[0699] C5 mRNA (Thermo assay ID Mm00439275_m1) and housekeeping gene GAPDH mRNA (Thermo assay ID Mm99999915_g1) in liver samples were analyzed using quantitative real-time PCR. The ΔΔCt method was used to calculate changes in C5 expression normalized to GAPDH relative to the saline control group.

[0700] ETX014 inhibits C5 mRNA expression in a dose-dependent manner. Figure 13 On day 14, a dose of 3 mg / kg achieved a reduction of more than 90% in C5 mRNA. The inhibition of C5 expression by ETX014 was durable, with a dose of 3 mg / kg per molecule showing significant knockdown of C5 mRNA until the end of the study on day 28.

[0701] For C5 protein level analysis, serum samples were measured using a commercially available C5 ELISA kit (Abcam ab264609). Serum C5 levels were calculated relative to the saline group mean at matched time points.

[0702] Serum protein data support mRNA analysis ( Figure 14 Treatment with ETX014 resulted in a dose-dependent decrease in serum C5 protein concentration. All doses of ETX014 reduced C5 protein levels by more than 70%, with the 3 mg / kg dose reducing C5 levels to almost undetectable levels on day 7 of the study. All doses maintained the reduction in serum C5 until the end of the study, with even the lowest dose of 0.3 mg / kg showing approximately 40% inhibition on day 28.

[0703] Example 4: NHP data of GalNAc-siRNA construct ETX023

[0704] ETX023 (targeting TTR mRNA) T1a reverse base-free

[0705] The pharmacology of ETX023 was evaluated in non-human primates (NHPs) by quantifying serum transthyretin (TTR) protein levels. A single subcutaneous dose of 1 mg / kg of GalNAc-conjugated siRNA ETX023 demonstrated durable inhibition of TTR protein expression.

[0706] Male cynomolgus macaques (3-5 years old, 2-3 kg) were randomly assigned to groups of 3 animals each. Animals were acclimatized for 2 weeks, and blood samples were collected 14 days prior to administration to provide baseline TTR concentrations. Liver biopsies were performed 18 or 38 days prior to administration to provide baseline mRNA levels. On day 0 of the study, animals received a single subcutaneous dose of 1 mg / kg GalNAc-siRNAETX023 dissolved in saline (sterile 0.9% sodium chloride). Liver biopsies were performed on days 3, 14, 28, 42, 56, 70, and 84 of the study, and RNA was extracted for TTR mRNA measurement. Blood samples were collected on days 1, 3, 7, 14, 28, 42, 56, 70, and 84 of the study for serum TTR concentration measurement and clinical blood chemistry analysis.

[0707] It was anticipated that inhibition of TTR mRNA expression would lead to a decrease in serum TTR protein levels. Serum TTR protein concentration was measured using a commercially available ELISA kit (Abcam ab231920). The TTR concentration for each individual animal was calculated as the day 1 fraction and plotted as the mean and standard deviation of the three animal groups. Figure 15 ).

[0708] A single dose of 1 mg / kg ETX023 resulted in a rapid and significant decrease in serum TTR concentration, reaching its lowest point at 28 days post-administration and maintaining inhibition until day 70.

[0709] Further data was obtained up to day 84. The same experiment was performed using ETX019, 021, and 025. Figure 16 , 17 84 days of data are provided in 18a and 19 (ETX019, 021, 023 and 025 respectively).

[0710] TTR mRNA was measured by real-time quantitative PCR using the TaqMan gene expression kit TTR (Thermo, assay ID Mf02799963_m1). GAPDH expression was also measured (Thermo, assay ID Mf04392546_g1) for reference. Relative TTR expression for each animal, normalized to GAPDH, was calculated relative to pre-dose levels using the DDCt method. A single 1 mg / kg dose of ETX023 also induced a rapid and significant decrease in hepatic TTR mRNA, reaching its lowest point at day 14 post-dose and maintaining inhibition until day 84. Figure 18b ).

[0711] Animal weight was measured weekly during the study. Fluctuations or decreases in weight were unrelated to ETX023 administration, and the animals continued to gain weight throughout the study. Figure 18c ).

[0712] Serum was analyzed within 2 hours using an automated biochemical analyzer. A significant increase in ALT (alanine aminotransferase) and AST (aspartate aminotransferase) is commonly used to demonstrate liver toxicity. Figure 18d ) or ALT ( Figure 18e The increase was not related to the administration of ETX023.

[0713] In a preferred aspect, the compounds of the present invention are capable of reducing serum protein levels of the target protein to below the initial (starting) concentration on day 0 for a period of at least about 14 days, at least about 21 days, at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, at least about 56 days, at least about 63 days, at least about 70 days, at least about 77 days, or at least about 84 days after day 0 (hereinafter referred to as the "dose duration"). "Day 0" as used herein refers to the day on which the compound of the present invention is first administered to the patient, in other words, the start of the dose duration or the time following administration.

[0714] In a preferred aspect, the compounds of the present invention are capable of reducing the serum protein level of the target protein to at least about 90% or less of the initial (starting) concentration on day 0, such as at least about 85% or less, at least about 80% or less, at least about 75% or less, at least about 70% or less, at least about 65% or less, at least about 60% or less, at least about 55% or less, at least about 50% or less, at least about 45% or less, at least about 40% or less, at least about 35% or less, at least about 30% or less, at least about 25% or less, at least about 20% or less, at least about 15% or less, at least about 10% or less, at least about 5% or less. Typically, this type of serum protein inhibition can be maintained for at least approximately 14 days after day 0, at least approximately 21 days after day 0, at least approximately 28 days after day 0, at least approximately 35 days after day 0, at least approximately 42 days after day 0, at least approximately 49 days after day 0, at least approximately 56 days after day 0, at least approximately 63 days after day 0, at least approximately 70 days after day 0, at least approximately 77 days after day 0, or at least approximately 84 days after day 0. More preferably, for a period of at least about 84 days after day 0, the serum concentration may be reduced to at least about 90% or less of the initial (starting) concentration on day 0, such as at least about 85% or less, at least about 80% or less, at least about 75% or less, at least about 70% or less, at least about 65% or less, at least about 60% or less, at least about 55% or less, at least about 50% or less, at least about 45% or less, at least about 40% or less.

[0715] In a preferred aspect, the compounds of the present invention are capable of achieving maximum inhibition of target protein serum protein levels to at least about 50% or less of the initial (starting) concentration on day 0, such as at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10%, or at least about 5%. Typically, such maximum inhibition of serum proteins occurs around day 14, around day 21, around day 28, around day 35, or around day 42 after day 0. More typically, such maximum inhibition of serum proteins occurs around day 14, around day 21, or around day 28 after day 0.

[0716] The specific compounds of this invention typically achieve maximum inhibition of serum protein levels of the target protein and / or inhibition over a period of at least about 84 days, as follows:

[0717] ETX019 typically achieves at least 50% inhibition of serum protein levels of the target protein (typically TTR) for approximately 7 to 21 days after day 0, particularly approximately 14 days after day 0, and / or typically maintains at least 90% inhibition of serum protein levels of the target protein (typically TTR) for a period of at least approximately 84 days after day 0 (as mentioned above, "day 0" as referred to herein means the day on which the compound of the invention was first administered to the patient and, by itself, indicates the time after administration).

[0718] ETX021 typically achieves at least 40% inhibition of serum protein levels of the target protein (typically TTR) from about 7 to 21 days after day 0, particularly about 14 days after day 0, and / or typically maintains at least 80% inhibition of serum protein levels of the target protein (typically TTR) for a period of at least about 84 days after day 0 (as mentioned above, "day 0" as referred to herein means the day on which the compound of the invention was first administered to the patient and, by itself, indicates the time after administration).

[0719] ETX023 typically achieves at least 20% inhibition of serum protein levels of the target protein (typically TTR) from about 7 to 21 days after day 0, particularly about 14 days after day 0, and / or typically maintains at least 50% inhibition of serum protein levels of the target protein (typically TTR) for a period of at least about 84 days after day 0 (as mentioned above, "day 0" in this document refers to the day on which the compound of the invention was first administered to the patient and, by itself, indicates the time after administration).

[0720] ETX025 typically achieves at least 50% inhibition of serum protein levels of the target protein (typically TTR) from approximately 7 to 21 days after day 0, particularly from approximately 14 days after day 0, and / or typically maintains at least 70% inhibition of serum protein levels of the target protein (typically TTR) for a period of at least approximately 84 days after day 0 (as mentioned above, "day 0" as referred to herein is the day on which the compound of the invention was first administered to the patient and per se indicates the time following administration). Suitable methods have been used to determine the inhibition of serum levels in non-human primates by delivering a single subcutaneous dose of the relevant active agent (e.g., ETX0023) dissolved in saline (sterile 0.9% sodium chloride). Suitable methods are described herein. It should be understood that this is not limiting, and other suitable methods with appropriate controls may be used.

[0721] Example 5 ETX023 (targeting TTR mRNA) T1a reverse abase-free

[0722] Total bilirubin levels remained stable throughout the study. Figure 22 )

[0723] Throughout the study, kidney health was monitored by assessing urea (blood urea nitrogen, BUN) and creatinine concentrations. Following a single 1 mg / kg dose of ETX023, both blood urea concentrations (BUN) and creatinine levels remained stable and within the expected range. Figure 23 and 24 ).

[0724] The scope of this invention is not limited to the specific embodiments disclosed, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such changes may be made without departing from the true scope and spirit of this disclosure and are intended to fall within its scope.

[0725] Another aspect of the invention is described below, with non-limiting examples illustrated in Figures 25-27 and Examples 6-15. The compounds described below are applicable to any aspect and embodiment disclosed above, for example in terms of use, nucleic acid length, definition, pharmaceutically acceptable compositions, administration, methods of inhibiting gene expression, and methods of treating or preventing diseases related to gene expression, unless otherwise readily apparent from this disclosure.

[0726] Figure 25A The three-antennae GalNAc (N-acetylgalactosamine) unit was described.

[0727] Figure 25B An alternative three-antennae GalNAc according to an embodiment of the invention is depicted, showing a change in the linking group.

[0728] Figure 26A The tri-antennae GalNAc-conjugated siRNA according to the present invention is depicted, showing the variation of the linker group.

[0729] Figure 26B A class of tri-antenna GalNAc-conjugated siRNAs according to an embodiment of the present invention is described.

[0730] Figure 26C A class of dual-antennae GalNAc-conjugated siRNAs according to an embodiment of the present invention is depicted, showing variations in the linker group.

[0731] Figure 26D A class of dual-antennae GalNAc-conjugated siRNAs according to another embodiment of the present invention is depicted, showing the variation of the linker group.

[0732] Figure 27A Another embodiment of a tri-antenna GalNAc-conjugated siRNA according to one embodiment of the present invention is described.

[0733] Figure 27B Depicting Figure 27A The variant shown has an alternative branched GalNAc conjugate.

[0734] Figure 27C A class of tri-antennae GalNAc-conjugated siRNAs according to an embodiment of the present invention is depicted, showing the variation of the linker group.

[0735] Figure 27D A class of dual-antennae GalNAc-conjugated siRNAs according to an embodiment of the present invention is depicted, showing variations in the linker group.

[0736] On the other hand, the forms of chemically modified RNAi reagents conjugated with ASGP-R ligands, as well as methods for the preparation and use of such conjugated molecules, are disclosed.

[0737] In some embodiments, the ASGP-R ligand comprises N-acetylgalactosamine (GalNAc). In some embodiments, the present invention provides siRNA conjugated to a triantennae or biantennae unit of GalNAc of formula (I):

[0738]

[0739] Formula I

[0740] In Equation I In this context, n is 0, 1, 2, 3, or 4. In some embodiments, the number of ethylene glycol units can vary independently of each other in different branches. For example, the middle branch can have n=4, while the side branches can have n=3, etc. Other embodiments may contain only two branches, as depicted in formula (II-a).

[0741]

[0742] Formula II -a

[0743] In Formula II and II In -a, n is selected from 0, 1, 2, 3, or 4. In some embodiments, the number of ethylene glycol units can vary independently of each other in different branches. For example, one branch may have n=4 or 3, while other branches may have n=3 or 2, and so on.

[0744] Additional GalNAc branches can also be added; for example, GalNAc units with 4-, 5-, 6-, 7-, 8-, and 9- branches can be used.

[0745] In relevant embodiments, the branched GalNAc can be chemically modified by adding another targeting moiety, such as lipids, cholesterol, steroids, bile acids, targeted (poly)peptides, including peptides and proteins (e.g., RGD peptide, transferrin, polyglutamic acid, polyaspartic acid, glycosylated peptides, biotin, desialylated glycoprotein insulin, and EGF).

[0746] Option 1. In a further embodiment, the GalNAc unit can be connected via a chain (such as formula (III)). The ligand shown in the diagram is attached to the RNAi reagent:

[0747]

[0748] Formula III

[0749] In Formula III In this case, m is selected from 0, 1, 2, 3, 4 or 5, and p is independent of m and is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and X is CH2 or O.

[0750] In another embodiment, the chain can be attached to the oligonucleotide via a phosphate (Z=O) or thiophosphate (Z=S) group, as in formula (IV). As shown in the image:

[0751]

[0752] Formula IV

[0753] These GalNAc branching units are preferably attached to the 3' or 5' end of the sense strand of the RNAi reagent via a specific ligand. In one embodiment, this is achieved via formula (V) The C6 amino linker shown is attached to the 3' junction of the RNAi reagent:

[0754]

[0755] Formula V

[0756] This connector is the starting point for synthesis as shown in Example 12.

[0757] The same connectors and chains as described above can be used with, as in Formula VI and VII The alternative branched GalNAc structures shown are used together:

[0758]

[0759] Style VI

[0760]

[0761] Equation VII

[0762] Similar to Formula II -a, based on VI and VII The biantennae form of the ligand can be used in the compositions of the present invention.

[0763] Option 2. In a further embodiment, the GalNAc unit can be connected via a chain (such as formula (III)). The ligand shown in -2) is attached to the RNAi reagent:

[0764]

[0765] Formula III -2

[0766] In Formula III In -2, q is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0767] In another embodiment, the chain can be attached to the oligonucleotide via a phosphate (Z=O) or thiophosphate (Z=S) group, as in formula (IV). As shown in the image:

[0768]

[0769] Formula IV

[0770] Such GalNAc branching units are preferably attached to the 3' or 5' end of the sense strand of a double-stranded RNAi reagent via a specific ligand. In one embodiment, attachment to the 3' end of the RNAi reagent is as shown in Example 14. In one embodiment, when the GalNAc ligand is attached to the 3' site, the transition link between the ligand and the 3' end of the oligonucleotide includes the formula (V... -a; see also . Figure 27C Alternatively, a suitable connector can be used, such as type (V). -b) shows the C6 amino linker:

[0771]

[0772] Formula V -a

[0773]

[0774] Formula V -b

[0775] Additional and / or alternative conjugation sites may include any non-terminal nucleotide, including sugar residues, phosphate groups, or nucleic acid bases.

[0776] The same connectors and chains can be used with, for example, VI. -2 and VII The alternative branched GalNAc structure shown in -2 can be used together:

[0777]

[0778] Style VI -2

[0779]

[0780] Equation VII -2

[0781] Features and chemical modifications of the RNAi reagent of the present invention

[0782] In some embodiments, the conjugated oligomeric compound (referred herein to as an RNA interference compound (RNAi compound)) comprises two strands, each strand having a sequence of 8 to 55 linked nucleotide monomer subunits (including inverted abase-free (ia) nucleotides) in either the antisense or sense strand. In some embodiments, the conjugated oligomeric compound strand comprises, for example, a sequence of 16 to 55, 53, 49, 40, 25, 24, 23, 21, 20, 19, 18, 17, or up to (about) 18-25, 18-23, 21-23 linked nucleotide monomer subunits. In some embodiments, the RNAi reagent of the present invention may have a hairpin structure having a single strand of the combined length of the two strands described above. (The term "nucleotide" as used throughout may also refer to a nucleoside (i.e., a nucleotide without a phosphate / thiophosphate group) when the context requires.)

[0783] In some embodiments, the double-stranded RNAi reagent is blunt-ended or has protrusions at one or both ends. In some embodiments, the protrusions are 1-6, 1-5, 1-4, 1-3, 2-4, 4, 3, 2, or 1 nucleotide of the antisense strand (at the 3' end or the 5' end) and 2-4, 3, 2, or 1 nucleotide of the sense strand (at the 3' end or the 5' end). In some exemplary embodiments, referring to Examples 6, constructs 6.1, 6.2, and 6.3, the RNAi reagent includes a 2-nucleotide protrusion at the 3' end of the antisense strand and a 2-nucleotide protrusion at the 3' end of the sense strand. In some other exemplary embodiments, referring to Examples 7, constructs 7.1 and 7.3; Examples 8, constructs 8.1 and 8.3; and Examples 9, constructs 9.1 and 9.3, the RNAi reagent includes a 2-nucleotide protrusion at the 3' end of the antisense strand and a blunt end at the other end. In some other exemplary embodiments, see Example 7, construct 7.3, where both ends of the construct are blunt. In another exemplary embodiment, see Example 9, construct 9.2, where the RNAi reagent includes a 4-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the other end.

[0784] In some embodiments, the construct is modified with a degradation-protective moiety that prevents or inhibits nuclease cleavage by using a terminal cap, one or more inverted abasic nucleotides, one or more phosphate-thiophosphate bonds, one or more deoxynucleotides (e.g., D-ribonucleotides, D-2'-deoxyribonucleotides, or other modified nucleotides), or combinations thereof. Such degradation-protective moieties may be present at any or all ends not conjugated to the ASGP-R ligand. In some embodiments, the degradation-protective moieties are alone or as any combination of the following groups: 1-4, 1-3, 1-2, or 1 phosphate-thiophosphate bond; 1-4, 1-3, 1-2, or 1 deoxynucleotide; and 1-4, 1-3, 1-2, or 1 inverted abasic nucleotide. In some exemplary embodiments, the degradation-protective moieties are configured as one of constructs 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.2, 8.3, 9.1, 9.2, and 9.3, as shown in Examples 6-15. Such exemplary protective portions can be used in conjunction with any RNAi reagent of the present invention.

[0785] In some embodiments, all or part of the ribose in the sense and / or antisense strands is modified. In some embodiments, at least 50%, 60%, 70%, 80%, 90% or more (e.g., 100%) of the ribose in the RNAi reagent is modified. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ribose are not modified.

[0786] In a preferred embodiment, the ribose modification includes a 2' substituent, such as a 2'-O-alkyl modification, including 2'-O-methyl and 2'-deoxyfluorine. Other modifications are known in the art, including 2'-deoxy, LNA (e.g., a 2'-O,4'-C methylene bridge or a 2'-O,4'-C ethylene bridge), 2'-methoxyethoxy (MOE), 2'-O-(CH2)OCH3, etc.

[0787] In some embodiments, many modifications provide different modification patterns, for example, as shown in the constructs in Examples 6-15, or as described in U.S. Patent Nos. 7,452,987; 7,528,188; 8,273,866; 9,150,606; and 10,266,825; all of which are incorporated herein by reference.

[0788] In some embodiments, the siRNA contains one or more heat-labile nucleotides, such as GNA, ENA, etc., for example at positions 11 (preferably), 12, 13 on the antisense strand and / or positions 9 and 10 (preferably) on the sense strand.

[0789] In addition, nucleic acid bases can be modified, for example, at the C4 position as described in U.S. Patent No. 10,119,136.

[0790] Generally, the RNAi reagents of the present invention target therapeutic targets, and inhibition of these targets will result in the prevention, mitigation, or treatment of diseases, including undesirable or pathological conditions. A large number of such targets are known in the art. Non-limiting examples of such targets include: ApoC, ApoB, ALAS1, TTR, GO, C5 (see Examples), etc. Generally, such targets are preferably expressed in the liver due to the abundant expression of ASGP-R on the surface of hepatocytes, but they can also be expressed in other tissues or organs. In a preferred embodiment, the target is human, and the RNAi reagent contains an antisense strand that is wholly or partially complementary to such a target. In some embodiments, the RNAi reagent may contain two or more chemically linked RNAi reagents targeting the same or different targets.

[0791] Example

[0792] Example 6: Reverse baseless chemistry using 5'-GalNAc

[0793] In all RNAi reagents described in the examples, the following conventions are used:

[0794] ia = reverse abase-free nucleotide;

[0795] m = 2'-O-methylnucleotide;

[0796] f = 2'-deoxy-2'-fluoronucleotide;

[0797] s = intermolecular bond between phosphate-thionucleotides;

[0798] Xd = 2'-deoxy-nucleotide;

[0799] ~ = chain.

[0800] Using standard synthesis techniques, various forms of the following constructs were synthesized, which have chains 1 and 2 and various tri-antennae GalNAc units according to the invention, as described above or Figures 26A-27B As shown in the image. 6.1.

[0802] (Up chain (sense(ss)): SEQ ID NO:1; Down chain (antonym) SEQ ID NO:2)

[0803] 6.2.

[0805] (Up chain (sense(ss)): SEQ ID NO:3; Down chain (antonym): SEQ ID NO:4)

[0806] 6.3.

[0808] (Up chain (sense(ss)): SEQ ID NO:5; Down chain (antonym): SEQ ID NO:6)

[0809]

[0810] Example 7: Reverse baseless chemistry using 3'-GalNAc

[0811] Using standard synthetic techniques, various forms of the following constructs were synthesized, which have chains 1 and 2 according to the invention and various tri-antennae GalNAc units according to the invention, as described above or Figures 26A-27B As shown in the example. To maintain consistency, the same sequence as in Example 6 is shown. 7.1

[0813] (Up chain (sense(ss)): SEQ ID NO:7; Down chain (antonym): SEQ ID NO:8)

[0814] 7.2

[0816] (Up chain (sense(ss)): SEQ ID NO:9; Down chain (antonym): SEQ ID NO:10)

[0817]

[0818] 7.3.

[0820] (Up chain (sense(ss)): SEQ ID NO:11; Down chain (antonym): SEQ ID NO:12)

[0821]

[0822] Example 8: Reverse baseless chemistry using 5'-GalNAc with alternative modification modes

[0823] Using standard synthetic techniques, various forms of the following constructs were synthesized, which have chains 1 and 2 according to the invention and various tri-antennae GalNAc units according to the invention, as described above or Figures 26A-27B As shown in the example. To maintain consistency, the same sequence as in Example 6 is shown here. 8.1.

[0825] (Up chain (sense(ss)): SEQ ID NO:13; Down chain (antonym): SEQ ID NO:14)

[0826] 8.2

[0828] (Up chain (sense(ss)): SEQ ID NO:15; Down chain (antonym): SEQ ID NO:16)

[0829]

[0830] 8.3.

[0832] (Up chain (sense(ss)): SEQ ID NO:17; Down chain (antonym): SEQ ID NO:18)

[0833]

[0834] Example 9: Reverse baseless chemistry using 5'-GalNAc with alternative modification modes

[0835] Using standard synthetic techniques, various forms of the following constructs were synthesized, which have chains 1 and 2 according to the invention and various tri-antennae GalNAc units according to the invention, as described above or Figures 26A-27B As shown in the example. To maintain consistency, the same sequence as in Example 6 is shown. 9.1.

[0837] (Up chain (sense(ss)): SEQ ID NO:19; Down chain (antonym): SEQ ID NO:20)

[0838] 9.2

[0840] (Up chain (sense(ss)): SEQ ID NO:21; Down chain (antonym): SEQ ID NO:22)

[0841] 9.3

[0843] (Up chain (sense(ss)): SEQ ID NO:23; Down chain (antonym): SEQ ID NO:24)

[0844]

[0845] Example 10: The baseline of siRNA-GalNAc conjugates

[0846] The constructs used in Example 6-15 are referenced by their numbers and listed in Table 12. Linkage 1 and Linkage 2 are shown in Figures 26 and 27, respectively.

[0847] Table 12.

[0848]

[0849] Table 13 below reflects the baseline achieved using various alternative constructs of the present invention.

[0850]

[0851]

[0852] In vitro pharmacodynamic characterization

[0853] The in vitro pharmacodynamic activities, binding affinity, and hepatic uptake of the eight constructs (GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc analogs) listed in Table 12 are based on clinically validated versions of these molecules.

[0854] Human liver cell line (HepG2 or Hep3B) transfection assay – Each of the GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc analogs was transfected at 37°C in human liver cell lines at 10 different concentrations in the presence of a transfection reagent (e.g., RNAiMAX).0 Incubation was performed at C for 0 and 24 hours. All incubations for each concentration were performed in quadruplicate. After incubation, each sample was lysed and the mRNA concentrations of HAO1 C5, TTR, and housekeeping genes (such as GAPDH) were analyzed by bDNA or RT-qPCR. The obtained mRNA concentration data were used to analyze and determine the silencing activity and IC50 of each of the GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc molecules. 50 .

[0855] Primary Human Hepatocyte Uptake Assay – This study evaluated the hepatic uptake and silencing activity of each of the GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc analogs in primary human hepatocytes. Each of the GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc analogs was administered at 37°C in primary human hepatocytes at 10 different concentrations. 0 Incubation was performed at C for 0, 4, and 72 hours. All incubations for each concentration were performed in quadruplicate. After incubation, each sample was lysed and the mRNA concentrations of HAO1, C5, TTR, and housekeeping genes (such as GAPDH) were analyzed by bDNA or RT-qPCR. The obtained mRNA concentration data were used to analyze and determine the silencing activity, uptake, and IC50 of each of the GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc molecules. 50 .

[0856] In vivo pharmacodynamic characterization

[0857] Following a single subcutaneous administration to male mice or cynomolgus monkeys, the in vivo pharmacodynamic activities of eight constructs (each of the GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc analogs) were compared with the in vivo pharmacodynamic activities of each of the clinically validated GO1 siRNA-GalNAc, C5 siRNA-GalNAc, and TTR siRNA-GalNAc molecules.

[0858] In vivo mouse pharmacology of each GO1 siRNA-GalNAc analogue was evaluated after a single subcutaneous dose of 0.3 mg / kg or 3 mg / kg as provided in Table 14 below. Two dose groups were established, in which each GO1 siRNA-GalNAc analogue was administered subcutaneously at 0.3 mg / kg or 1 mg / kg to male C57BL / 6 mice (n=3 / time point / group). Blood samples were collected at different time points to obtain serum and liver biopsy samples to determine serum glycolic acid concentration by LCMS and HAO1 mRNA concentration by RT-qPCR or bDNA assay. Animals from each group were sacrificed at each specific time point, and blood (approximately 0.5 mL / animal) and liver (approximately 100 mg) were collected. For groups 1 through 9, blood (approximately 0.5 mL / animal) and liver (approximately 100 mg) were collected from 3 animals / time points / groups at 24, 48, 96, 168, 336, 504, and 672 hours post-administration. Group 10 (n=3) was the control group, which was not administered the drug to provide baseline values ​​for serum glycolic acid and HAO1 mRNA concentrations. The pharmacodynamic effects of increased serum glycolic acid and silencing of HAO1 mRNA in the liver at different time points after administration were compared with those of the Group 10 control group in serum and liver samples.

[0859] Table 14. Pharmacological study design of GO1 siRNA-GalNAc analog in mice

[0860]

[0861] Example 11: Using click chemistry 5' conjugation (Option 1)

[0862]

[0863] In this embodiment, the sense chain of oligonucleotide 101 was synthesized on a solid support and coupled with commercially available octynephosphatamide 102 to obtain the desired oligonucleotide on the solid support using a click chemistry precursor. This yielded pure oligonucleotide 103 after standard cleavage and deprotection. Azide 104 was dissolved in DMSO (150 µL / mg), and this solution was added to 10 OD oligonucleotide 103 in 100 µL of water. The reaction mixture was then incubated overnight at room temperature. The conjugated oligonucleotide 105 was desalted on Glen Gel-Pak™ to remove organic matter and the acetoxy protecting group was removed by treatment with methylamine, followed by preparative HPLC to obtain pure oligonucleotide 106, which was annealed with an equimolar amount of the sense chain to obtain the final duplex.

[0864] Example 12: 5' join (Option 2)

[0865]

[0866] In this embodiment, the sense strand of oligonucleotide 101 was synthesized on a solid support and coupled with commercially available phosphoramide 108 to obtain the desired oligonucleotide on a solid support. This yielded pure oligonucleotide 109 after standard cleavage and deprotection. Amine 109 was dissolved in water (15 µL / OD), and this solution was added to a solution of acid 110 in DMSO (100 mL / mg), followed by the addition of 10 molar equivalents of EDC and 10 molar equivalents of HOBT, and the reaction mixture was incubated overnight at room temperature. The conjugated oligonucleotide 111 was then desalted on Glen Gel-Pak™ to remove organic matter and the acetoxy protecting group was removed by treatment with methylamine, followed by preparative HPLC to obtain pure oligonucleotide 112, which was annealed together with an equimolar amount of the sense strand to obtain the final doublet.

[0867]

[0868] Example 13: Using click chemistry 5' conjugation (Option 1)

[0869]

[0870] For the synthesis of oligonucleotide construct 119, a similar approach was used, in which the tri-antennae GalNAc conjugate was loaded onto a solid support 118 (CPG) and oligonucleotide synthesis was performed. After cleavage and deprotection, purification yielded pure oligonucleotide 119, which was annealed together with the antisense strand to provide the final double strand in the desired pure form. In another approach, the synthesis of the 3' conjugate is also similar to that of 116, starting with an amino-linked oligonucleotide 113 and post-synthetic conjugation with GalNAc carboxylic acid to obtain the conjugated oligonucleotide 119.

[0871] Example 14: 3' join (Option 2)

[0872]

[0873] For the synthesis of oligonucleotide construct 119, a similar approach was used, in which the tri-antennae GalNAc conjugate was loaded onto a solid support 118 (CPG) and oligonucleotide synthesis was performed. Purification after cleavage and deprotection provided pure oligonucleotide 119, which was annealed together with the antisense strand to provide the final double strand in the desired pure form. In another approach, the synthesis of the 3' conjugate is also similar to that of 116, starting with an amino-linked oligonucleotide 113 and conjugating it with GalNAc carboxylic acid post-synthesis to obtain the conjugated oligonucleotide 119.

[0874] Example 15: Composition of suffixes

[0875]

[0876]

[0877] In this method, the synthesis of the 3' conjugate is also similar to that of 116, starting with an amino-linked oligonucleotide 113 and postsynthetically conjugated with GalNAc carboxylic acid to obtain the conjugated oligonucleotide 121, which is annealed together with the antisense strand to obtain the final doublet in the desired pure form.

[0878] The preceding examples are not intended to be limiting. Based on this disclosure, those skilled in the art will understand that many changes can be made to the specific materials and disclosure without departing from the spirit and scope of the invention and still obtaining the same or similar results.

[0879] statement

[0880] 1. A modified RNAi reagent comprising an RNA interference compound (RNAi compound) conjugated to an ASGP-R ligand via a ligand, wherein the ligand comprises:

[0881]

[0882] (Formula III) )

[0883] Where m is selected from 0, 1, 2, 3, 4 or 5, and p is independent of m and is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; and X is selected from O and CH2.

[0884] 2. The modified RNAi reagent according to Statement 1, wherein m=1.

[0885] 3. The modified RNAi reagent according to Statement 1, wherein x is O.

[0886] 4. The modified RNAi reagent according to Statement 1, wherein p=6.

[0887] 5. The modified RNAi reagent according to Statement 1, wherein m=1, p=6, and x is 0.

[0888] 6. The modified RNAi reagent according to Statement 1, wherein the ASGP-R ligand comprises branched GalNAc.

[0889] 7. The modified RNAi reagent according to Statement 6, wherein the branched GalNAc is selected from the group consisting of:

[0890]

[0891] Formula I

[0892] and

[0893]

[0894] Formula II -a

[0895] Where n is 0, 1, 2, 3 or 4.

[0896] 8. The modified RNAi reagent according to Statement 7, wherein n=1.

[0897] 9. The modified RNAi reagent according to Statement 6, wherein the branched GalNAc contains

[0898]

[0899] Style VI ,

[0900] Or in the form of two tentacles.

[0901] 10. The modified RNAi reagent according to Statement 6, wherein the branched GalNAc comprises or is in the form of a biantennae.

[0902]

[0903] Equation VII ,

[0904] Or in the form of two tentacles.

[0905] 11. The modified RNAi reagent according to Statement 1, wherein the co-strand is attached to the 5' end of the sense strand.

[0906] 12. The modified RNAi reagent according to Statement 11, wherein the lineage is as shown in Formula IV. The attachment shown.

[0907] ;

[0908] Where Z is either P or S.

[0909] 13. The modified RNAi reagent according to Statement 11, wherein the co-strand is attached to the 3' end of the sense strand.

[0910] 14. The modified RNAi reagent according to Statement 13, wherein the lineage is as shown in Formula V -a or V -b indicates the attachment:

[0911]

[0912] Formula V -a

[0913]

[0914] Formula V -b

[0915] 15. The modified RNAi reagent according to Statement 1, which is as follows: Figure 26A , 26B As shown in 26C or 26D.

[0916] 16. The modified RNAi reagent according to Statement 15, wherein the RNAi compound contains a modified ribose modified at the 2' position.

[0917] 17. The modified RNAi reagent according to Statement 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluorine, and 2'-deoxy.

[0918] 18. The modified RNAi reagent according to Statement 1, wherein the RNAi compound contains one or more degradation-protected moieties at any or all ends not conjugated to the ASGP-R ligand.

[0919] 19. The modified RNAi reagent according to Statement 18, wherein the degradation-protecting portion is alone or as a selection of any combination of the following: 1-4 phosphate-thiophosphate bonds, 1-4 deoxynucleotides and 1-4 inverse abase-free nucleotides.

[0920] 20. The modified RNAi reagent according to Statement 19, wherein the degradation-protecting portion is selected from the configurations present in one of the following constructs: 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.2, 8.3, 9.1, 9.2 and 9.3.

[0921] 21. A modified RNAi reagent comprising an RNA interference compound (RNAi compound) conjugated to an ASGP-R ligand via a ligand, wherein the ligand comprises:

[0922]

[0923] (Formula III) -2)

[0924] Where q is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0925] 22. The modified RNAi reagent according to statement 21, wherein q=1.

[0926] 23. The modified RNAi reagent according to statement 21, wherein the ASGP-R ligand comprises branched GalNAc.

[0927] 24. The modified RNAi reagent according to statement 23, wherein the branched GalNAc is selected from the group consisting of:

[0928]

[0929] Formula I

[0930] and

[0931]

[0932] Formula II -a

[0933] Where n is 0, 1, 2, 3 or 4.

[0934] 25. The modified RNAi reagent according to statement 24, wherein n=1.

[0935] 26. The modified RNAi reagent according to statement 23, wherein the branched GalNAc contains

[0936]

[0937] Style VI ,

[0938] Or in the form of two tentacles.

[0939] 27. The modified RNAi reagent according to statement 23, wherein the branched GalNAc comprises or is in the form of a biantennae.

[0940]

[0941] Equation VII ,

[0942] Or in the form of two tentacles.

[0943] 28. The modified RNAi reagent according to statement 21, wherein the lineage strand is attached to the 5' end of the sense strand.

[0944] 29. The modified RNAi reagent according to statement 28, wherein the lineage is as shown in formula IV. The attachment shown.

[0945] ;

[0946] Where Z is either P or S.

[0947] 30. The modified RNAi reagent according to Statement 28, wherein the lineage strand is attached to the 3' end of the sense strand.

[0948] 31. The modified RNAi reagent according to statement 30, wherein the lineage is as shown in formula V -a or V -b indicates the attachment:

[0949]

[0950] Formula V -a

[0951]

[0952] Formula V -b

[0953] 32. The modified RNAi reagent according to statement 31, which, as Figure 27A , 27B As shown in 27C or 27D.

[0954] 33. The modified RNAi reagent according to statement 21, wherein the RNAi compound contains a modified ribose modified at the 2' position.

[0955] 34. The modified RNAi reagent according to statement 33, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluorine, and 2'-deoxy.

[0956] 35. The modified RNAi reagent according to Statement 21, wherein the siRNA contains one or more degradation-protected moieties at any or all ends not conjugated to the ASGP-R ligand.

[0957] 36. The modified RNAi reagent according to statement 35, wherein the degradation-protecting portion is alone or as a choice of any combination of the following: 1-4 phosphate-thiophosphate bonds, 1-4 deoxynucleotides and 1-4 inverse abase-free nucleotides.

[0958] 37. The modified RNAi reagent according to statement 36, wherein the degradation-protecting portion is selected from the configurations present in one of the following constructs: 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.2, 8.3, 9.1, 9.2 and 9.3.

[0959] 39. A method for preparing an RNAi reagent according to statement 1 or 2, the method being shown in Examples 11-15.

[0960] 40. A method for preventing, alleviating, or treating a disease in a subject, the method comprising administering to the subject a therapeutic amount of an RNAi reagent according to statement 1 or 2 that is effective in preventing, alleviating, or treating the disease, thereby preventing, alleviating, or treating the disease.

[0961] 41. The method according to statement 40, wherein the subject is a human being.

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969]

[0970]

[0971]

[0972]

[0973]

[0974]

[0975]

[0976]

[0977]

[0978]

[0979]

[0980]

[0981]

[0982]

[0983]

[0984]

[0985]

[0986]

[0987]

[0988]

[0989]

[0990]

[0991]

Claims

1. A compound comprising the following structure: Formula (I) in: R1 is independently selected from the group consisting of the following items each time it appears: hydrogen, methyl, and ethyl; X1 and X2 are independently selected from the group consisting of the following items each time they appear: methylene, oxygen, and sulfur; m is an integer from 1 to 6; n is an integer from 1 to 10; q, r, s, t, v are independent integers from 0 to 4, provided that: (i) q and r cannot both be 0 at the same time; and (ii) s, t, and v cannot all be 0 at the same time; Z is the oligonucleotide moiety, represented by the following structure: in: Z1, Z2, Z3, and Z4 are independently oxygen or sulfur each time they appear; and One of the bonds between P and Z2 and between P and Z3 is a single bond, and the other bond is a double bond; R2 is a hydroxyl group. and of formula (I) is in: A1 is hydrogen; a is an integer of 2 or 3; and b is an integer from 2 to 5.

2. The compound according to claim 1, wherein R1 is hydrogen each time it appears.

3. The compound according to claim 1, wherein m = 3.

4. The compound according to claim 1, wherein n = 6.

5. The compound according to claim 1, wherein X1 is oxygen and X2 is methylene.

6. The compound according to claim 5, wherein: q = 1， r = 2， s = 1， t = 1， v = 1。 7. The compound according to claim 1, wherein both X1 and X2 are methylene groups.

8. The compound according to claim 7, wherein: q = 1， r = 3， s = 1， t = 1， v = 1。 9. The compound of claim 1, wherein the oligonucleotide is an RNA compound capable of modulating the expression of a target gene; and wherein, The RNA compound comprises an RNA duplex comprising a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, and the second strand is at least partially complementary to the first strand, and wherein each of the first strand and the second strand has a 5' end and a 3' end.

10. The compound of claim 9, wherein the oligonucleotide is an RNA compound capable of inhibiting the expression of the target gene.

11. The compound of claim 1, wherein, The compound comprises the structure of formula (III): Equation (III) Or include the structure of the following formula (V): Formula (V) in as shown in formula (III) or formula (V) is in: A1 is hydrogen; a is an integer of 2 or 3; and b is an integer from 2 to 5.

12. The compound of claim 1, wherein, The compound is selected from: Formula (IX) Formula (XI).

13. The compound according to any one of claims 1 to 12, wherein the oligonucleotide further comprises one or more degradation-protected portions at one or more ends, wherein the one or more degradation-protected portions are selected from phosphate-thionucleotide inter-linked bonds, diphosphate-thionucleotide inter-linked bonds, and reverse abase-free nucleotides, wherein the reverse abase-free nucleotide is present at the distal end of the chain carrying the ligand portion.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier, diluent or excipient.

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