expression and / or functional inhibitors

Abstract

The present application relates to inhibitors and compositions containing the inhibitors, and their use in the treatment or prevention of metabolic diseases or conditions, for example metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity and / or diseases or conditions associated with lipogenesis.

Description

Technical Field

[0001] This application provides inhibitors suitable for therapeutic use, such as nucleic acid compounds, like siRNA. Furthermore, this application provides methods for preparing these compounds, and methods for using such compounds to treat various diseases and conditions. Background Technology

[0002] Inhibitors, such as oligonucleotides, are inhibitors of gene expression and / or the expression or function of other targets (e.g., LNCRNAs), and 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 / oligonucleotides that prevent protein formation through gene silencing.

[0003] In particular, over the past two decades, a variety of modified siRNA compounds have been developed for diagnostic and therapeutic purposes, including siRNA / RNAi therapeutics for treating a wide range of diseases, including central nervous system diseases, inflammatory diseases, metabolic disorders, tumors, infectious diseases, and eye diseases.

[0004] This application relates to inhibitors, such as oligomers (e.g., nucleic acids, such as oligonucleotides / oligonucleotide compounds), and their use in the treatment and / or prevention of diseases.

[0005] The SLC25A5 gene belongs to the ANT gene family, which itself is a superfamily containing genes encoding brown adipose mitochondrial uncoupling proteins and mitochondrial phosphotransporters. This gene is a member of the mitochondrial carrier subfamily of solute carrier protein genes. The product of this gene is adenine nucleotide transporter 2 (ANT2). ANT2 is a major component of the mitochondrial permeability transition pore complex, which catalyzes the exchange of mitochondrial ATP with cytoplasmic ADP. Due to its antitransport function, ANT2 maintains mitochondrial membrane potential by regulating the ADP / ATP ratio during oxidative phosphorylation. When ANT2 is acylated by SIRT4, it promotes the uncoupling of mitochondrial membrane potential. Although membrane potential uncoupling usually leads to apoptosis, ANT2 has been found to have anti-apoptotic effects. Therefore, it is hypothesized to be a component of MMS19-XPD, mediating TFIIH-dependent DNA damage responses. Summary of the Invention

[0006] This application relates to the following as defined in the claims, and in particular to the following: In one aspect, this application relates to an inhibitor of SLC25A5 / ANT2 expression and / or function, wherein the inhibitor is coupled to one or more ligand moieties.

[0007] On the other hand, this application relates to an inhibitor according to this application, wherein the inhibitor is a siRNA oligomer.

[0008] On the other hand, this application relates to inhibitors of SLC25A5 / ANT2 expression and / or function, wherein the inhibitor is a siRNA oligomer.

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

[0010] In another aspect, this application relates to an inhibitor according to this application, wherein the one or more ligand portions comprise one or more GalNAc ligands or comprise one or more GalNAc ligand derivatives.

[0011] In another aspect, this application relates to an inhibitor according to this application, wherein the one or more ligand portions comprise one or more GalNAc ligand derivatives.

[0012] On the other hand, this application relates to an inhibitor according to this application, wherein the target of said inhibitor is SLC25A5 / ANT2.

[0013] On the other hand, this application relates to an inhibitor according to this application, wherein the inhibitor is a nucleic acid for inhibiting SLC25A5 expression, the nucleic acid comprising a double-stranded segment comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) At least partially complementary to a portion of the RNA transcribed from the SLC25A5 gene, and (ii) Contains at least 17 consecutive nucleosides that differ from any of the first-strand sequences listed in Table 2 by 0 or 1 nucleoside.

[0014] On the other hand, this application relates to an inhibitor according to this application, wherein the inhibitor is a nucleic acid for inhibiting SLC25A5 expression, the nucleic acid comprising a double-stranded segment comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) At least partially complementary to a portion of the RNA transcribed from the SLC25A5 gene, and (ii) Contains at least 17 consecutive nucleosides that differ from any of the first-strand modification sequences listed in Table 3 by 0 or 1 nucleoside.

[0015] On the other hand, this application relates to an inhibitor according to this application, wherein the first chain comprises nucleoside 2-18 of any sequence as defined in claim 8 or 9, and in particular wherein the first chain comprises nucleoside 2-18 of any sequence as defined in Table 2 or 3.

[0016] On the other hand, this application relates to an inhibitor according to this application, wherein the second chain comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any second chain sequence listed in Table 2 by 0 or 1 nucleoside, and wherein the second chain has a region that is at least 85% complementary to the first chain on the 17 consecutive nucleosides.

[0017] On the other hand, this application relates to an inhibitor according to this application, wherein the second chain comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any of the second sequences listed in Table 4 by 0 or 1 nucleoside, and wherein the second chain has a region that is at least 85% complementary to the first chain on the 17 consecutive nucleosides.

[0018] On the other hand, this application relates to an inhibitor of this application, wherein the first chain comprises any of the first chain sequences listed in Table 2.

[0019] On the other hand, this application relates to an inhibitor of this application, wherein the first chain comprises any of the first chain modification sequences listed in Table 3.

[0020] On the other hand, this application relates to an inhibitor of this application, wherein the second chain comprises any of the second chain sequences listed in Table 2.

[0021] On the other hand, this application relates to an inhibitor of this application, wherein the second chain comprises any of the first chain modification sequences listed in Table 4.

[0022] In another aspect, this application relates to an inhibitor of this application, wherein the first chain comprises any of the following sequences: SEQ ID NO:304, SEQ ID NO:323, SEQ ID NO:439, SEQ ID NO:453 and SEQ ID NO:496.

[0023] On the other hand, this application relates to an inhibitor according to this application, wherein the first chain comprises SEQ ID NO:304 or SEQ ID NO:280.

[0024] On the other hand, this application relates to an inhibitor according to this application, wherein the first chain comprises any of the following sequences: SEQ ID NO:856, SEQ ID NO:875, SEQ ID NO:991, SEQ ID NO:1005 and SEQ ID NO:1048.

[0025] In another aspect, this application relates to an inhibitor of this application, wherein the first chain comprises SEQ ID NO: 856 or SEQ ID NO: 832.

[0026] In another aspect, this application relates to an inhibitor of this application, wherein the second chain comprises any of the following sequences: SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772.

[0027] On the other hand, this application relates to an inhibitor of this application, wherein the second chain comprises SEQ ID NO:580 or SEQ ID NO:556.

[0028] In another aspect, this application relates to an inhibitor of this application, wherein the second chain comprises any of the following sequences: SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324.

[0029] On the other hand, this application relates to an inhibitor of this application, wherein the second chain comprises SEQ ID NO:1132 or SEQ ID NO:1108.

[0030] On the other hand, this application relates to an inhibitor according to this application, comprising a first chain and a second chain, wherein the first chain and the second chain comprise a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside, a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside, or substantially a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside:

[0031] On the other hand, this application relates to an inhibitor according to this application, comprising first and second chains, said first and second chains comprising a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside:

[0032] On the other hand, this application relates to an inhibitor according to this application, comprising first and second chains, said first and second chains comprising a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside:

[0033] On the other hand, this application relates to an inhibitor according to this application, comprising first and second chains, said first and second chains comprising a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside:

[0034] On the other hand, this application relates to an inhibitor according to this application, comprising first and second chains, said first and second chains comprising a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside:

[0035] On the other hand, this application relates to an inhibitor according to this application, comprising first and second chains, said first and second chains comprising a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any of the following combinations of the first and second sequences by 0 or 1 nucleoside:

[0036] On the other hand, this application relates to an inhibitor according to this application, which is an siRNA oligomer having a first strand and a second strand, wherein: i) The first strand of the siRNA has a length of 15 to 30 nucleotides, preferably 19 to 25 nucleotides, more preferably 23 or 25 nucleotides, and even more preferably 23 nucleotides; and / or ii) The second strand of the siRNA has a length of 15 to 30 nucleotides, preferably 19 to 25 nucleotides, and more preferably 21 nucleotides.

[0037] On the other hand, this application relates to an inhibitor according to this application, wherein the length of the first chain is 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides.

[0038] On the other hand, this application relates to an inhibitor according to this application, wherein the length of the second chain is 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19, 21 or 23 nucleosides.

[0039] On the other hand, this application relates to an inhibitor of this application, wherein the length of the double-stranded segment of the nucleic acid is 17 to 30 nucleotides, more preferably 19, 21 or 23 nucleotides.

[0040] On the other hand, this application relates to an inhibitor of this application, wherein the complementary region between the first strand and the RNA portion transcribed from the SLC25A5 gene has a length of 17 to 30 nucleotides.

[0041] On the other hand, this application relates to an inhibitor of this application, wherein the nucleic acid further comprises one or more single-stranded nucleoside overhangs, optionally wherein the overhangs are present on a first or second strand, preferably at the 3' end of the first or second strand, and / or wherein the overhangs comprise 1 to 4 nucleosides, more preferably 2 nucleosides.

[0042] On the other hand, this application relates to an inhibitor of this application, wherein the nucleic acid is an siRNA oligonucleotide.

[0043] On the other hand, this application relates to an inhibitor according to this application, wherein the second sense chain further comprises one or more abasic nucleosides in the terminal region of the second chain, and wherein the abasic nucleosides are linked to adjacent nucleosides via reverse nucleoside internucleotide bonds.

[0044] On the other hand, this application relates to an inhibitor according to this application, wherein the second chain comprises: i) Two or more abase-free nucleotides in the terminal region of the second strand; and / or ii) Two or more abase-free nucleotides in the 5' or 3' terminal region of the second strand; and / or iii) Two or more abasic nucleosides in the 5' or 3' terminal region of the second strand, wherein said abasic nucleosides are present in the overhangs as described herein; and / or iv) Two or more consecutive abasic nucleoside chains in the terminal region of the second chain, wherein preferably one of such abasic nucleosides is a terminal nucleoside; and / or v) Two or more consecutive abasic nucleotides in the 5' or 3' terminal region of the second strand, wherein preferably one of such abasic nucleotides is a terminal nucleotide in the 5' or 3' terminal region of the second strand; and / or vi) Linking at least one base-free nucleotide in the terminal region of the second chain to the reverse internucleotide bond of an adjacent basic nucleotide; and / or vii) Linking at least one base-free nucleotide from the 5' or 3' terminal region of the second strand to the reverse internucleotide bond of an adjacent basic nucleotide; and / or viii) A base-free nucleoside that is the penultimate nucleoside, which is linked via a reverse bond to a non-terminal nucleoside (referred to herein as the penultimate nucleoside); and / or ix) When the chain is read toward the ends, the baseless nucleosides are linked by 5'-3' bonds as two terminal nucleosides; x) When the chain is read in the direction of the end containing the terminal nucleoside, the baseless nucleoside is connected by 3'-5' bonds as two terminal nucleosides; xi) is a nucleoside with no bases at the two ends, wherein the penultimate nucleoside is linked to the penultimate nucleoside by a reverse bond, and wherein the reverse bond is a 5-5' reverse bond or a 3'-3' reverse bond; xii) As a nucleoside with no bases at the two terminal positions, wherein the penultimate nucleoside is linked to the penultimate nucleoside via a reverse bond, and wherein... (1) When read toward the end containing the terminal and the penultimate anucleotide, the reverse bond is a 5-5' reverse bond, and the bond between the terminal and the penultimate anucleotide is a 3'5' bond; or (2) When read toward the end containing the end and the penultimate anucleotide, the reverse bond is a 3-3' reverse bond and the bond between the end and the penultimate anucleotide is 5'3'.

[0045] On the other hand, this application relates to an inhibitor according to this application, wherein the reverse nucleoside internucleotide bond is located at the distal end of the 5' terminal region of the second chain, or at the distal end of the 3' terminal region of the second chain.

[0046] On the other hand, this application relates to an inhibitor according to this application, wherein the reverse nucleoside inter-bond is a 3'3 reverse bond.

[0047] On the other hand, this application relates to an inhibitor according to this application, wherein the reverse nucleoside inter-bond is a 5'5 reverse bond.

[0048] On the other hand, this application relates to an inhibitor according to this application, wherein the second sense chain further comprises one or more abasic nucleosides in the terminal region of the second chain, and wherein the abasic nucleosides are linked to adjacent nucleosides via reverse nucleoside internucleotide bonds.

[0049] On the other hand, this application relates to an inhibitor according to this application, wherein the second chain comprises two consecutive abasic nucleosides in its 5' terminal region, one of which is the terminal nucleoside of the 5' terminal region of the second chain, and the other abasic nucleoside is the penultimate nucleoside of the 5' terminal region of the second chain, wherein: (a) The penultimate abasic nucleoside is connected to the adjacent first basic nucleoside in the adjacent 5' proximal region via a reverse nucleoside inter-bond; (b) The reverse key is a 5-5' reverse key; and (c) When read toward the end containing the terminal and the penultimate abase-free nucleotide, the bond between the terminal and the penultimate abase-free nucleotide is 3'5'.

[0050] The inhibitor according to this application, or the inhibitor for the purpose described in this application, wherein the reverse nucleoside internucleotide bond is located at the distal end of the 5' terminal region of the second strand, or at the distal end of the 3' terminal region of the second strand.

[0051] On the other hand, this application relates to an inhibitor according to this application, wherein (i) The first and second strands each have a length of 23 nucleotides; (ii) Two thiophosphate nucleoside bonds are located at three consecutive positions in the 5' proximal region of the second chain, wherein the first thiophosphate nucleoside bond is located between the adjacent first basic nucleoside of (a) and the adjacent second basic nucleoside in the 5' proximal region of the second chain, and the second thiophosphate nucleoside bond is located between the adjacent second basic nucleoside and the adjacent third basic nucleoside in the 5' proximal region of the second chain. (iii) Two thiophosphate nucleoside bonds are respectively located at three consecutive positions in the 5' and 3' end regions of the first chain, thereby connecting the terminal nucleosides in the 5' and 3' end regions of the first chain to the corresponding penultimate 5' and 3' nucleosides via thiophosphate nucleoside bonds, and each first penultimate 5' and 3' nucleoside is connected to the corresponding penultimate 5' and 3' nucleoside via thiophosphate nucleoside bonds; and (iv) The second strand of the nucleic acid is directly or indirectly coupled to one or more ligand portions of the 3' terminal region of the second strand.

[0052] On the other hand, this application relates to an inhibitor according to this application, wherein two consecutive inverted abasic nucleosides in the 5' terminal region of the second chain are present as the following 5' terminal motif: in: T represents 2'Me ribose modification. B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of the second strand. Z represents the remaining 19 consecutive basic nucleosides in the second chain.

[0053] In another aspect, this application relates to an inhibitor according to this application, wherein one or more nucleosides on the first and / or second chains are modified to form modified nucleosides.

[0054] In another aspect, this application relates to an inhibitor according to this application, wherein the modification is a modification at the 2'-OH group of the ribose, optionally selected from 2'-Me modification or 2'-F modification.

[0055] In another aspect, this application relates to an inhibitor according to this application, wherein the first chain includes 2'-F at any position of position 14, position 2, position 6 or any combination thereof, counting from position 1 of the first chain.

[0056] In another aspect, this application relates to an inhibitor according to this application, wherein the second chain comprises a 2'-F modification at positions 7 and / or 9, and / or positions 11 and / or 13, counting from position 1 of the second chain.

[0057] In another aspect, this application relates to an inhibitor according to this application, wherein the first chain and the second chain each contain a 2'-Me modification and a 2'-F modification.

[0058] In another aspect, this application relates to an inhibitor according to this application, which is siRNA, wherein the siRNA comprises at least one thermally unstable modification suitably at one or more positions 1 to 9 on the first strand, starting from position 1, and / or at one or more positions on the second strand aligned with positions 1 to 9 on the first strand, wherein the unstable modification is selected from modified nonlocked nucleic acids (UNA) and glycol nucleic acids (GNA), preferably glycol nucleic acids.

[0059] In another aspect, this application relates to an inhibitor according to this application, wherein, starting from position 1 of the first strand, the siRNA contains at least one thermally unstable modification at position 7 of the first strand.

[0060] In another aspect, this application relates to an inhibitor according to this application, which is siRNA, wherein, starting from position 1 of the second strand, the siRNA contains three or more 2'-F modifications at positions 7 to 13 of the second strand, for example, four, five, six or seven 2'-F modifications at positions 7 to 13 of the second strand.

[0061] In another aspect, this application relates to an inhibitor according to this application, which is siRNA, wherein, counting from position 1 of the second strand, the second strand contains at least three, for example four, five or six, 2'-Me modifications at positions 1 to 6 of the second strand.

[0062] In another aspect, this application relates to an inhibitor according to this application, which is siRNA, wherein the first strand contains at least five consecutive 2'-Me modifications in the 3' end region, preferably including the terminal nucleoside of the 3' end region, or at least within one or two nucleosides of the terminal nucleoside at a distance from the 3' end region.

[0063] In another aspect, this application relates to an inhibitor according to this application, which is siRNA, wherein the first strand contains seven consecutive 2'-Me modifications in the 3' end region, preferably including terminal nucleosides in the 3' end region.

[0064] In another aspect, this application relates to an inhibitor according to this application, which is a siRNA oligomer, wherein the siRNA oligomer further comprises one or more thiophosphate nucleoside internucleotide bonds.

[0065] In another aspect, this application relates to an inhibitor according to this application, wherein the one or more thiophosphate nucleoside internucleotide bonds are located between at least three consecutive positions in a 5' or 3' proximal terminal region of the second chain, wherein the proximal terminal region is preferably adjacent to the terminal region, wherein the positions of the one or more abase-free nucleosides of the second chain are as defined herein.

[0066] In another aspect, this application relates to an inhibitor according to this application, wherein one or more thiophosphate nucleoside interbonds are located between at least three consecutive positions in the 5' and / or 3' end regions of the first chain, wherein, preferably, the end positions of the 5' and / or 3' end regions of the first chain are connected to their adjacent positions by thiophosphate nucleoside interbonds.

[0067] In another aspect, this application relates to an inhibitor according to this application, wherein the oligomer is siRNA, and the second strand of the siRNA is directly or indirectly coupled to one or more ligand moieties, wherein the ligand moieties are generally present in the terminal region of the second strand, preferably in its 3' terminal region.

[0068] In another aspect, this application relates to an inhibitor according to this application, wherein the ligand portion comprises i) One or more GalNAc ligands; and / or ii) One or more GalNAc ligand derivatives; and / or iii) One or more GalNAc ligands and / or GalNAc ligand derivatives conjugated to the siRNA via a linker.

[0069] In another aspect, this application relates to an inhibitor according to this application, wherein one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly coupled to the 5' or 3' terminal region of the second strand of the siRNA oligomer, preferably coupled to its 3' terminal region.

[0070] In another aspect, this application relates to an inhibitor according to this application, wherein the ligand portion comprises In another aspect, this application relates to an inhibitor according to this application, which comprises the following structure: in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X1 and X2 are each selected independently from the following group: 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; and (ii) s, t, and v cannot all be 0 at the same time; Z represents the oligonucleotide moiety.

[0071] In another aspect, this application relates to an inhibitor according to this application, which comprises the following structure Oligonucleotides represent consecutive nucleosides in the second chain.

[0072] In another aspect, this application relates to an inhibitor according to this application, which comprises the following structure in: r and s are independent integers selected from 1 to 16; and Z represents the oligonucleotide moiety.

[0073] In another aspect, this application relates to an inhibitor according to this application, which comprises the following structure Oligonucleotides represent consecutive nucleosides in the second chain.

[0074] In another aspect, this application relates to an inhibitor according to this application, wherein said structure is coupled to the 3' terminal region of the second chain.

[0075] In another aspect, this application relates to an inhibitor according to this application, which is formulated into a pharmaceutical composition together with excipients and / or a carrier.

[0076] In another aspect, this application relates to pharmaceutical compositions comprising a combination of an inhibitor of one or more of the foregoing aspects and a pharmaceutically acceptable excipient or carrier.

[0077] In another aspect, this application relates to pharmaceutical compositions according to this application, which further comprise a GLP-1 agonist and / or a THR-β agonist.

[0078] In another aspect, this application relates to pharmaceutical compositions according to this application, wherein the GLP-1 agonist is a GLP-1 / GIP dual agonist, a GLP-1 / FGF21 dual agonist, a GLP-1 / GCGR dual agonist, or a GLP-1 / GIP / GCGR triple agonist.

[0079] In another aspect, this application relates to a pharmaceutical composition according to this application, wherein the GLP-1 agonist is semaglutide.

[0080] In another aspect, this application relates to a pharmaceutical composition according to this application, wherein the THR-β agonist is resmetirom.

[0081] In another aspect, this application relates to a pharmaceutical composition according to this application, wherein the GLP-1 / GIP dual agonist is tirzepatide.

[0082] In another aspect, this application relates to a pharmaceutical composition according to this application, further comprising one or more of the following: an amylin receptor agonist (e.g., pramlintide), and / or an amylin + calcitonin dual receptor agonist, and / or a glucagon receptor agonist, and / or an FXR receptor agonist (e.g., cilofexor or obeticholic acid). FGF-21 analogs or FGF-21 receptor agonists (e.g., efruxifermin), and / or FGF-19 analogs or FGF-19 receptor agonists (e.g., aldafermin), and / or galactoglobulin 3 inhibitors (e.g., belapectin), and / or PPARα agonists (e.g., elafibrinor), and / or PPARγ agonists (e.g., pioglitazone or rosiglitazone), and / or mixed PPARα and / or δ and / or γ agonists, and / or pan-PPARαγδ agonists (e.g., laniline). Acetyl-CoA desaturase activators, and / or ASK1 inhibitors (e.g., selonsertib), and / or LOXL2 inhibitors (e.g., simtuzumab), and / or dual CCR2 / 5 inhibitors (e.g., cenicriviroc), and / or inhibitors of enzymes in the de novo lipogenesis (DNL) pathway, including citrate / isocitrate carrier (CIC), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), and / or inhibitors of enzymes in the cholesterol biosynthesis pathway (e.g., HMGCoA reductase inhibitors, such as atorvastatin).

[0083] In another aspect, this application relates to an inhibitor or a pharmaceutical composition according to this application for use in therapy.

[0084] In another aspect, this application relates to an inhibitor or pharmaceutical composition according to this application for the prevention and / or treatment of metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or diseases or conditions associated with lipogenesis, and / or for the purpose of reducing lipogenesis.

[0085] In another aspect, this application relates to an inhibitor or a pharmaceutical composition for the use described in this application, wherein the inhibitor or the pharmaceutical composition is used in combination with a GLP-1 agonist and / or a THR-β agonist.

[0086] In another aspect, this application relates to an inhibitor or a pharmaceutical composition for the use described in this application, wherein the GLP-1 agonist is a GLP-1 / GIP dual agonist, a GLP-1 / FGF21 dual agonist, a GLP-1 / GCGR dual agonist, or a GLP-1 / GIP / GCGR triple agonist.

[0087] In another aspect, this application relates to an inhibitor or a pharmaceutical composition for the use described in this application, wherein the GLP-1 agonist is semaglutide.

[0088] In another aspect, this application relates to an inhibitor or a pharmaceutical composition for the use described in this application, wherein the THR-β agonist is retinoic acid.

[0089] In another aspect, this application relates to an inhibitor or a pharmaceutical composition for the use described in this application, wherein the GLP-1 / GIP dual agonist is tesipatide.

[0090] In another aspect, this application relates to inhibitors or pharmaceutical compositions for the purposes described in this application, wherein said inhibitors or pharmaceutical compositions will be used in combination with one or more of the following: an amylin receptor agonist (e.g., pramlintide), and / or an amylin + calcitonin dual receptor agonist, and / or a glucagon receptor agonist, and / or an FXR receptor agonist (e.g., ciprofoxetine or obeticholic acid), and / or an FGF-21 analog or an FGF-21 receptor agonist (e.g., iluxivmin), and / or an FGF-19 analog or an FGF-19 receptor agonist (e.g., odavmin), and / or a galactoglobulin 3 inhibitor (e.g., berapetin), and / or a PPARα agonist (e.g., erafibrano), and / or a PPARγ agonist. (e.g., pioglitazone or rosiglitazone), and / or mixed PPARα and / or δ and / or γ agonists, and / or pan-PPARαγδ agonists (e.g., lanilano), and / or acetyl-CoA desaturase activators, and / or ASK1 inhibitors (e.g., seloselti), and / or LOXL2 inhibitors (e.g., simtuzumab), and / or dual CCR2 / 5 inhibitors (e.g., sinivirol), and / or inhibitors of enzymes in the de novo lipogenesis (DNL) pathway, including citrate / isocitrate carriers (CIC), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and / or inhibitors of enzymes in cholesterol biosynthesis pathways (e.g., HMGCoA reductase inhibitors, such as atorvastatin).

[0091] In another aspect, this application relates to the use of SLC25A5 / ANT2 as a target for identifying one or more therapeutic agents for the prevention and / or treatment of metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity and / or diseases or conditions associated with lipogenesis, and / or for reducing lipogenesis.

[0092] In another aspect, this application relates to a method for treating or preventing metabolic diseases or conditions such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis and / or a method for reducing lipogenesis, comprising administering to a patient an inhibitor of SLC25A5 / ANT2 expression and / or function, such as the inhibitor according to this application.

[0093] In another aspect, this application relates to a method according to this application, wherein an inhibitor of SLC25A5 / ANT2 expression and / or function is administered together with a GLP-1 agonist and / or a THR-β agonist.

[0094] In another aspect, this application relates to a method according to this application, wherein the GLP-1 agonist is a GLP-1 / GIP dual agonist, a GLP-1 / FGF21 dual agonist, a GLP-1 / GCGR dual agonist, or a GLP-1 / GIP / GCGR triple agonist.

[0095] In another aspect, this application relates to a method according to this application, wherein the GLP-1 agonist is semaglutide.

[0096] In another aspect, this application relates to a method according to this application, wherein the GLP-1 / GIP dual agonist is tesipatide.

[0097] In another aspect, this application relates to a method according to this application, wherein the THR-β agonist is remetidine.

[0098] In another aspect, this application relates to a method according to this application, wherein an inhibitor of SLC25A5 / ANT2 expression and / or function is administered together with one or more of the following: an amylin receptor agonist (e.g., pramlintide), and / or an amylin + calcitonin dual receptor agonist, and / or a glucagon receptor agonist, and / or an FXR receptor agonist (e.g., ciprofloxacin or obeticholic acid), and / or an FGF-21 analog or an FGF-21 receptor agonist (e.g., iluxivmin), and / or an FGF-19 analog or an FGF-19 receptor agonist (e.g., odavmin), and / or a galactoglobulin 3 inhibitor (e.g., belapetin), and / or a PPARα agonist (e.g., erafibrano), and / or a PPARγ agonist (…). Examples include pioglitazone or rosiglitazone, and / or mixed α and / or δ and / or γ agonists, and / or pan-PPARαγδ agonists (e.g., lanilano), and / or acetyl-CoA desaturase activators, and / or ASK1 inhibitors (e.g., seloselti), and / or LOXL2 inhibitors (e.g., simtuzumab), and / or dual CCR2 / 5 inhibitors (e.g., sinivirol), and / or enzyme inhibitors in the de novo lipogenesis (DNL) pathway, including citrate / isocitrate carrier (CIC), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), and / or enzyme inhibitors in the cholesterol biosynthesis pathway (e.g., HMG-CoA reductase inhibitors, such as atorvastatin).

[0099] In another aspect, this application relates to the use of the inhibitor or pharmaceutical composition according to this application in the preparation of a medicament for treating metabolic diseases or conditions such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis, and / or for reducing lipogenesis.

[0100] In another aspect, this application relates to the use of SLC25A5 / ANT2 as a biomarker for metabolic diseases or conditions such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis.

[0101] In another aspect, this application relates to an in vivo method for using SLC25A5 / ANT2 to predict susceptibility to metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis, typically by monitoring the sequence and / or expression level and / or function of SLC25A5 / ANT2 in samples obtained from patients.

[0102] In another aspect, this application relates to a method for predicting a patient's susceptibility to metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis, said method comprising: (a) Obtaining samples from patients (b) Detect the sequence and / or expression level and / or function of SLC25A5 / ANT2 in the samples obtained from the patient. (c) Based on the sequence and / or expression and / or function of SLC25A5 / ANT2 in samples obtained from patients, predict susceptibility to metabolic diseases or conditions associated with diseases or conditions such as non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or diseases or conditions associated with lipogenesis. (d) It is preferable to administer an effective amount of the SLC25A5 / ANT2 inhibitor to the confirmed patient. Attached Figure Description

[0103] Figure 1a : An exemplary linear configuration of a coupling.

[0104] Figure 1b : An exemplary branching configuration of a coupling.

[0105] Figure 2 : Suitable for the connector and ligand portions of the construct used according to this application, including tether 1a. Although Figure 2 Linkers coupled with oligonucleotides are described, but it should be understood that this application also covers conjugates of the same linkers with oligonucleotides as disclosed herein.

[0106] It should also be understood that, although Figure 2 Described based on Figure 2The product molecule, consisting of a linker and ligand moiety described herein, is linked to the oligonucleotide moiety. This product may also comprise or consist essentially of the following molecules, wherein the linker and ligand moiety are substantially as follows: Figure 2 As shown, it is linked to the oligonucleotide moiety, but as Figure 2 The F substituent on the cyclooctyl ring shown is replaced by a substituent that can occur via hydrolytic substitution, such as an OH substituent, or the OH substituent itself can synthesize a linker. Thus, the (a) chain 1a construct can be essentially composed of components having, as shown in the diagram... Figure 2 The molecular composition of the linker and ligand moieties shown has an F substituent on the cyclooctyl ring; or (b) the chain 1a construct can be substantially composed of molecules having, as shown in the diagram. Figure 2 The molecular composition of the connector and ligand motifs shown is, but as... Figure 2 The F substituent on the cyclooctyl ring shown is replaced by an OH substituent, or (c) the chain 1a construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0107] Figure 3 : Suitable for the connector and ligand portions of the construct used according to this application, including tether 1b. Although Figure 3 Linkers coupled with oligonucleotides are described, but it should be understood that this application also covers conjugates of the same linkers disclosed herein with oligonucleotides.

[0108] about Figure 2 The explanations and Figure 2 The possibility that the F substituent on the cyclooctyl ring shown can be replaced by other substituents (which can occur via hydrolytic substitution, such as an OH substituent, or the OH substituent itself can synthesize a linker) also applies to the chain 1b construct. Thus, (a) the chain 1b construct can be substantially composed of components having, as shown in the diagram, Figure 3 The shown linker and ligand moieties have an F substituent on the cyclooctyl ring; or (b) the chain 1b construct can be substantially composed of molecules having, as shown in the diagram. Figure 3 The molecular composition of the connector and ligand motifs shown is, but as... Figure 3 The F substituent on the cyclooctyl ring shown is replaced by an OH substituent, or (c) the chain 1b construct may comprise a mixture of molecules as defined in (a) and / or (b).

[0109] Figure 4 : Suitable for the connector and ligand portions of the construct used according to this application, including tether 2a. Although Figure 4 Linkers coupled with oligonucleotides are described, but it should be understood that this application also covers conjugates of the same linkers disclosed herein with oligonucleotides.

[0110] Figure 5 : Suitable for the connector and ligand portions of the construct used according to this application, including tether 2b. Although Figure 5 Linkers coupled with oligonucleotides are described, but it should be understood that this application also covers conjugates of the same linkers disclosed herein with oligonucleotides.

[0111] Figure 6 The structure described in statements numbered 1-101 disclosed in this document.

[0112] Figure 7 The structure described in Clauses 1-56 disclosed herein.

[0113] Figure 8a and 8b : A reverse-base-free construct that can be used with the nucleic acid sequence of this application as described herein. For Figure 8a The GalNAc connector is connected to the 5' end region of the used ligature. Figure 8a (Not shown in the text). For Figure 8b The GalNAc connector is connected to the 3' end region of the prosthetic chain used. Figure 8b (Not shown in the image).

[0114] like Figure 8a The iaia shown in the 3' end region of the positive chain represents: (i) the two abasic nucleotides of the penultimate and terminal nucleotides in the 3' end region of the positive chain, (ii) wherein the 3'-3' reverse bond is between the penultimate nucleotide of the positive chain (i.e., position 21 of the positive chain, where position 1 is the terminal 5' nucleotide of the positive chain) and the adjacent penultimate abasic residue, and (iii) the bond between the terminal and penultimate abasic nucleotide is 5'-3' when read toward the 3' end region containing the terminal and penultimate abasic nucleotides.

[0115] like Figure 8b The iaia shown in the 5' end region of the positive chain indicates: (i) two abasic nucleotides in the penultimate nucleotide and the terminal nucleotide in the 5' end region of the positive chain, (ii) where the 5'-5' reverse bond is between the penultimate nucleotide of the positive chain (i.e., at position 1 of the positive chain, excluding the iaia motif in the 5' end region of the positive chain in the nucleotide position numbering on the positive chain) and the adjacent penultimate abasic residue, and (iii) when read toward the 5' end region containing the terminal and penultimate abasic nucleotides, the bond between the terminal and penultimate abasic nucleotides is 3'-5'.

[0116] Figure 9a and 9b Based on the double-stranded constructs in Table 5.

[0117] Figure 10Summary of the knockdown effects of single-dose GalNAc-siRNA, ETX-M00001397, ETX-M00001570, ETX-M00001378, ETX-M00001513, and ETX-M00001527 (1 mg / kg or 3 mg / kg) on ​​mRNA and protein in mouse liver tissue. Non-target-specific siRNA was used as a negative control. The y-axis values ​​represent relative mRNA or protein expression levels, and data have been normalized to a saline control group (n=4). Each data point represents a relative mRNA or protein expression level, and data are expressed as mean ± SEM. Experimental sample n=4.

[0118] Figure 11 NAFLD activity scoring (NAS) was performed on H&E-stained liver samples using the clinical criteria outlined by Kleiner et al. (2005). Total NAS represented the sum of scores for steatosis, inflammation, and ballooning degeneration, ranging from 0 to 8. NAS scores were determined by Gubra using the Gubra Histopathological Objective Scoring Technology (GHOST) deep learning application developed by Gubra with VIS software (Visiopharm, Denmark) to more accurately and objectively stage the disease in the DIO-NASH mouse model. Results were presented as changes (improvement or deterioration) in NAS scores compared to pre-drug biopsy at the end of the study. The percentage of animals showing at least a 1 or 2-point improvement in NAS was also shown.

[0119] Figure 12 ALT and AST levels in plasma samples after 12 weeks of treatment with ETX-312 (ETX-M00001378) were measured using a commercial kit (Roche Diagnostics) on a cobas c501 automated analyzer. ALT and AST levels were elevated in DIO-NASH mice (solvent sc, siCtrl, solvent PO). Treatment with ETX-312 (ETX-M00001378) alone or in combination with semaglutide or retemetiride significantly reduced ALT and AST levels. Results are expressed as absolute values, and data are presented as mean ± SEM. The experimental sample size was n=16. * p<0.05; *** p<0.001; **** p<0.0001.

[0120] Figure 13TIMP-1 and PIIINP are non-invasive blood biomarkers for NAFLD / NASH and predict liver fibrosis. TIMP-1 in plasma collected from EDTA tubes was measured using a commercially available ELISA kit (R&D Systems). PIIINP in plasma collected from EDTA tubes was measured using a commercially available ELISA kit (Cusabio). TIMP-1 and PIIINP levels were elevated in DIO-NASH mice (solvent sc, siCtrl, solvent PO). Treatment with ETX-312 alone (ETX-M00001378) or in combination with semaglutide or remetrol significantly reduced TIMP-1 and PIIINP levels. Results are expressed in absolute values, and data are presented as mean ± SEM. The experimental sample size was n=16. Outlier analysis was performed by comparing the studentized residuals of the linear model fitted to the subcutaneous treatment subset data with the critical Bonferroni α level (0.05 / 88 = ~0.00057). One animal from the smegglutinin group was identified as an anomalous sample and validated by influence analysis. This animal was considered highly anomalous and influential, and was therefore excluded from all TIMP-1 and PIIINP analyses. * p<0.05; *** p<0.001; **** p<0.0001.

[0121] Figure 14 The final liver weight to body weight ratio indicated hepatomegaly in DIO-NASH mice. Treatment with ETX-312 alone (ETX-M00001378) or in combination with semaglutide or retemetiride significantly reduced the liver weight to body weight ratio. Results are expressed as liver weight:body weight percentage, and data are presented as mean ± SEM. The experimental sample size was n=16. ** p<0.01; **** p<0.0001.

[0122] Figure 15 Overview of human hydrodynamic injection (HDI) mouse studies.

[0123] Figure 16 Knockdown of human SLC25A5 mRNA in a hydrodynamically controlled injection (HDI) mouse model. Mice received subcutaneous siRNA (3 mg / kg) on ​​day -6, followed by HDI of the human SLC25A5 plasmid on day 0. qPCR on day 1 confirmed decreased expression of human SLC25A5 mRNA, with several siRNAs, including ETX-M00001378 (ETX-M00001751) and ETX-M00001354 (ETX-M00002666), showing the most significant knockdown compared to the control group. Data are presented as mean ± SEM. Experimental sample n=5. *** p<0.001; **** p<0.0001.

[0124] Figure 17 Overview of a single-dose 84-day study in non-human primates (NHP).

[0125] Figure 18 Hepatic SLC25A5 mRNA knockdown and ANT2 protein reduction in NHP treated with ETX-M00001378 (ETX-M00001751) and ETX-M00001354 (ETX-M00002666). Non-treatment-naïve cynomolgus monkeys received a single subcutaneous dose of 3 mg / kg ETX siRNA, with liver biopsies performed on days -7, 28, 56, and 84. Treatment resulted in a reduction of up to 72% in hepatic SLC25A5 mRNA and 57-60% in ANT2 protein, with a sustained reduction of approximately 40% in ANT2 levels up to day 84. Data are presented as mean ± SEM. Syringe icons indicate a single subcutaneous dose of 3 mg / kg.

[0126] Figure 19 Tolerance of ETX-M00001378 (ETX-M00001751) and ETX-M00001354 (ETX-M00002666) in NHP. Hepatic function plasma markers (ALT, AST, ALP, GGT) were assessed before treatment (day 0) and at days 14, 28, 56, and 84 after treatment. All markers remained within the normal range, indicating good tolerability. ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; GGT: gamma-glutamyl transferase. Dashed lines indicate the reference range of normal values. Detailed Implementation

[0127] This application specifically provides inhibitors, such as oligomers like nucleic acids, such as repressive RNA molecules (which may be referred to as iRNA or siRNA), and compositions containing such inhibitors, which can affect the expression of a target, for example, by binding to mRNA transcribed from a gene. The target may be located intracellularly, such as in the cells of a subject (e.g., a human). These inhibitors can be used to prevent and / or treat medical conditions associated with, for example, the expression of a target gene.

[0128] Specifically, this application identifies inhibitors of SLC25A5 / ANT2 expression and / or function that can be used to prevent and / or treat metabolic diseases or conditions, such as those associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or those associated with lipogenesis.

[0129] ADP / ATP translocase 2 (ANT2) is produced in humans by a translocase on the X chromosome. SLC25A5A gene-encoded protein. This protein acts as a reverse transporter for ADP / ATP exchange between the mitochondrial matrix and the cytoplasm.

[0130] This application relates to inhibitors of SLC25A5 / ANT2 expression and / or function. Therefore, in some embodiments, this application relates to inhibitors of SLC25A5 gene expression, such as those targeting [specific genes]. SLC25A5 siRNA of mRNA transcribed from a gene. In some embodiments, this application relates to an inhibitor of the function of the gene product ANT2. Both options are included when references are made herein to an inhibitor of SLC25A5 / ANT2 or an inhibitor of this application.

[0131] In humans, ANT2 is composed of SLC25A5 The gene (SEQ ID NO:1381) encodes...

[0132] SEQ ID NO:1381(SLC25A5): The inventors employed a network analysis approach, which enabled them to assign multiple genes or proteins to a smaller number of driving processes and mine these processes to identify influential drug targets. This method leverages information often overlooked in standard gene set analyses—interactions between known and predicted genes (and proteins) and the inclusion of other genes in the same or related pathways. Specifically, the inventors used network models to analyze a genome-wide association study (GWAS) meta-analysis of nonalcoholic fatty liver disease (NAFLD), which highlighted SLC25A5 / ANT2 as a preferred target for NAFLD among other known targets associated with NAFLD.

[0133] The inhibition disclosed herein can target the gene SLC25A5 or the protein ANT2 caused by the expression of the SLC25A5 gene, and when SLC25A5 / ANT2 is mentioned, it explicitly includes the inhibition of gene expression or function, as well as the inhibition of protein products.

[0134] definition The term "first strand," also referred to herein as the antisense strand or guide strand, and used interchangeably herein, refers to a nucleic acid strand, such as the strand of siRNA (e.g., dsiRNA), that contains a region substantially complementary to a target sequence (e.g., mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., the target sequence). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur in the interior or terminal regions of the molecule. In some embodiments, the double-stranded nucleic acid of this application, such as siRNA formulations, contains nucleotide mismatches in the antisense strand.

[0135] The term “second strand” (also referred to herein as the sense strand or the passerby strand, which are used interchangeably) refers to a strand of nucleic acid (e.g., siRNA) that contains regions substantially complementary to the antisense strand region, as defined herein.

[0136] In the context of molecules containing nucleic acids having a ligand portion (optionally also having a linker portion), the nucleic acid of this application may be referred to as an oligonucleotide portion or oligonucleotide portion.

[0137] Oligonucleotides are short nucleic acid polymers. While oligonucleotides contain phosphodiester bonds between their nucleoside components (bases plus sugar), this application is not limited to oligonucleotides where adjacent nucleosides are always linked by such phosphodiester bonds, but also considers other nucleoside oligomers linked by bonds other than phosphate bonds. For example, the bond between nucleotides can be a thiophosphate bond. Therefore, the term "oligonucleotide" herein encompasses oligonucleotides and other nucleoside oligomers. According to this application, oligonucleotides are preferred, which are nucleic acids that are at least partially oligonucleotides. According to this application, oligonucleotides having one or more or most of a phosphodiester backbone bond between nucleosides are also preferred. According to this application, oligonucleotides having one or more or most of a phosphodiester backbone bond between nucleosides and also having one or more phosphodiester backbone bonds between nucleosides (typically in the terminal regions of the first and / or second chains) are also preferred.

[0138] In this document, the nucleic acid is preferably a double-stranded oligonucleotide containing one or more phosphate thioester backbone bonds between the nucleosides. Therefore, in all instances where oligonucleotides are mentioned in this application, particularly in the chemical structures disclosed herein, the oligonucleotide can also be an oligonucleotide as defined herein.

[0139] In some embodiments, the double-stranded nucleic acid (e.g., siRNA) of this application contains a nucleoside mismatch in the positive strand. In some embodiments, the nucleoside mismatch is, for example, within 5, 4, 3, 2, or 1 nucleoside segments from the 3' end of the nucleic acid (e.g., siRNA).

[0140] In another implementation, the nucleoside mismatch occurs, for example, at the 3' end nucleoside of a nucleic acid (e.g., siRNA).

[0141] "Target sequence" (also known as target RNA or target mRNA) refers to the continuous portion of the nucleoside sequence of an mRNA molecule formed during gene transcription, including the mRNA of the product of RNA processing of the primary transcription product, or the continuous portion of the nucleotide sequence of any RNA molecule (e.g., LNCRNA that needs to be repressed).

[0142] The target sequence can be approximately 10-35 nucleotides long, for example, approximately 15-30 nucleotides. For example, the target sequence length can be approximately 15-30 nucleotides, 15-29 nucleotides, 15-28 nucleotides, 15-27 nucleotides, 15-26 nucleotides, 15-25 nucleotides, 15-24 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, or 15-18 nucleotides. Nucleosides, 15-17 nucleosides, 18-30 nucleosides, 18-29 nucleosides, 18-28 nucleosides, 18-27 nucleosides, 18-26 nucleosides, 18-25 nucleosides, 18-24 nucleosides, 18-23 nucleosides, 18-22 nucleosides, 18-21 nucleosides, 18-20 nucleosides, 19-30 nucleosides, 19-29 nucleosides 19-28 nucleosides, 19-27 nucleosides, 19-26 nucleosides, 19-25 nucleosides, 19-24 nucleosides, 19-23 nucleosides, 19-22 nucleosides, 19-21 nucleosides, 19-20 nucleosides, 20-30 nucleosides, 20-29 nucleosides, 20-28 nucleosides, 20-27 nucleosides, 20-26 nucleosides 20-25 nucleosides, 20-24 nucleosides, 20-23 nucleosides, 20-22 nucleosides, 20-21 nucleosides, 21-30 nucleosides, 21-29 nucleosides, 21-28 nucleosides, 21-27 nucleosides, 21-26 nucleosides, 21-25 nucleosides, 21-24 nucleosides, 21-23 nucleosides, or 21-22 nucleosides. Ranges and lengths falling between the above ranges and lengths are also considered part of this application.

[0143] The terms “ribonucleoside” or “nucleoside” can also refer to modified nucleosides, as will be discussed further below.

[0144] Nucleic acids can be DNA or RNA, and can contain modified nucleosides. RNA is the preferred nucleic acid.

[0145] The terms “iRNA,” “siRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used interchangeably in this document and refer to reagents containing RNA that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. siRNA guides sequence-specific degradation of mRNA through RNA interference (RNAi).

[0146] Double-stranded RNA, referred to herein as a "double-stranded siRNA (dsiRNA) agent," "double-stranded siRNA (dsiRNA) molecule," "double-stranded RNA (dsRNA) agent," "double-stranded RNA (dsRNA) molecule," "dsiRNA agent," "dsiRNA molecule," or "dsiRNA," is a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, exhibiting "sense" and "antisense" orientations relative to the target RNA. Most of the nucleosides in each strand of the nucleic acid (e.g., dsRNA molecule) are preferably ribonucleosides; however, in this case, each or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides or modified ribonucleosides. Furthermore, the term "siRNA" as used herein may comprise chemically modified ribonucleosides.

[0147] The term "modified nucleoside" refers to a nucleoside that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Therefore, the term modified nucleoside encompasses substitution, addition, or removal of, for example, functional groups or atoms, from internucleotide bonds, sugar moieties, or nucleobases. For the purposes of this specification and claims, any such modification (such as that used in siRNA-type molecules) is covered by "iRNA," "RNAi agent," "siRNA," or "siRNA agent."

[0148] The length of the double-stranded segment of the nucleic acid, such as dsRNA, in this application can be approximately 9 to 40 base pairs, for example 9 to 36 base pairs, for example approximately 15 to 30 base pairs, for example approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs, for example approximately 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18- 27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs.

[0149] The two strands that form a double helix structure can be different parts of a larger molecule or separate molecules, such as an RNA molecule.

[0150] The term "nucleoside overhang" refers to at least one unpaired nucleoside extending from the double-stranded structure of a double-stranded nucleic acid. The double-stranded nucleic acid may contain an overhang of at least one nucleoside; or, the overhang may contain at least two, three, four, five, or more nucleosides. The nucleoside overhang may contain or consist of nucleoside analogs (including deoxynucleosides). The overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleoside of the overhang may be at the 5' end, 3' end, or both ends of the antisense or sense strand.

[0151] In some embodiments, the antisense strand has 1-10 nucleosides at the 3' or 5' end, such as 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside overhangs.

[0152] "Flat-ended" or "flat-ended" refers to a double-stranded nucleic acid that has no unpaired nucleosides at its ends, i.e., no nucleoside overhangs. The nucleic acids in this application include nucleic acids that have no nucleoside overhangs at one end or neither end.

[0153] Unless otherwise stated, the term "complementarity," when used to describe the relationship between a first nucleoside sequence and a second nucleoside sequence, refers to the ability of an oligonucleotide containing the first nucleoside sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleoside sequence under certain conditions and form a double-stranded structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press").

[0154] Complementary sequences within nucleic acids (e.g., dsiRNA) described herein include base pairings of an oligonucleotide or polynucleotide containing a first nucleoside sequence with an oligonucleotide or polynucleotide containing a second nucleoside sequence over the entire length of one or both nucleoside sequences. Such sequences may be referred to herein as “completely complementary” to each other. However, when the first and second sequences are referred to herein as “substantially complementary” or “partially complementary,” the two sequences may be completely complementary, or they may form one or more mismatched base pairs, such as 2, 4, or 5 mismatched base pairs, but preferably no more than 5, while retaining the ability to hybridize under conditions most relevant to their final application, such as repressing gene expression via a RISC pathway. In determining complementarity, overhangs should not be considered mismatches. For example, a nucleic acid (e.g., double-stranded RNA) containing one 17-nucleoside oligonucleotide and another 19-nucleoside oligonucleotide, wherein the longer oligonucleotide contains a 17-nucleoside sequence that is completely complementary to the shorter oligonucleotide, may still be referred to as “completely complementary.”

[0155] As used herein, “complementary” sequences may also include non-Watson-Crick base pairs or base pairs formed from or entirely of non-natural and modified nucleosides, provided they meet the above requirements regarding hybridization ability. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairing or Hoogstein base pairing.

[0156] The terms “complementary,” “fully complementary,” and “substantially / partially complementary” used in this article may refer to base pairing between the sense and antisense strands of a nucleic acid (e.g., dsiRNA), or between the antisense strand and the target sequence of a double-stranded nucleic acid (e.g., siRNA).

[0157] In this application, the second strand of the nucleic acid, particularly the dsiRNA for inhibiting SLC25A5, is at least partially complementary to the first strand of the nucleic acid. In some embodiments, partial complementarity is defined as follows: the length of the double-stranded segment formed by the first and second strands of the nucleic acid is at least 17 base pairs, and it contains no more than 1, 2, 3, 4, or 5 mismatched base pairs.

[0158] In some embodiments, if the length of the double-stranded segment formed by the first and second strands of the nucleic acid of this application is 19 base pairs and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs, then they are partially complementary. In some embodiments, if the length of the double-stranded body formed by the first and second strands of the nucleic acid of this application is 21 base pairs and contains no more than 1, 2, 3, 4, or 5 mismatched base pairs, then they are partially complementary.

[0159] Alternatively, if the length of the double helix formed by the first and second strands of the nucleic acid in this application is at least 17 base pairs, of which at least 14, 15, 16 or 17 of the base pairs are complementary base pairs, particularly Watson-Crick base pairs, then the two strands are partially complementary.

[0160] In some embodiments, if the length of the double-stranded segment formed by the first and second strands of the nucleic acid of this application is 19 base pairs, wherein at least 14, 15, 16, 17, 18, or all 19 base pairs are complementary base pairs, particularly Watson-Crick base pairs, then the two strands are partially complementary. In some embodiments, if the length of the double-stranded segment formed by the first and second strands of the nucleic acid of this application is 21 base pairs, wherein at least 16, 17, 18, 19, 20, or all 21 base pairs are complementary base pairs, particularly Watson-Crick base pairs, then the two strands are partially complementary.

[0161] As used herein, a nucleic acid that is “substantially complementary” or “partially complementary” to at least a portion of messenger RNA (mRNA) refers to a polynucleotide that is substantially or partially complementary to a continuous portion of a target mRNA (e.g., the mRNA encoding a gene). In some embodiments, the continuous portion of the mRNA is any of the sequences listed in Table 1, i.e., SEQ ID NO: 1-276. For example, if a polynucleotide sequence is substantially or partially complementary to an uninterrupted portion of the mRNA encoding a target gene, then the polynucleotide is complementary to at least a portion of the mRNA of that target gene.

[0162] Therefore, in some preferred embodiments, the antisense oligonucleotides disclosed herein are completely complementary to the target gene sequence.

[0163] In other embodiments, the antisense oligonucleotides disclosed herein are substantially or partially complementary to the target RNA sequence and comprise a continuous nucleoside sequence that is at least about 80% complementary to the equivalent region of the target RNA sequence over its entire length, for example, at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary or 100% complementary.

[0164] In some implementations, the first strand (antisense strand) of the nucleic acid in this application is transcribed from... SLC25A5 The continuous portions of the gene's RNA are partially or completely complementary. In some embodiments, the first strand of the nucleic acid in this application is... SLC25A5 At least 17 consecutive nucleotides of the mRNA are partially or completely complementary. In some embodiments, the first strand of the nucleic acid of this application is... SLC25A5The mRNA is partially or completely complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleotides. In some embodiments, the first strand of the nucleic acid of this application is partially or completely complementary to a continuous portion of 17, 18, 19, 20, 21, 22, or 23 nucleotides of any sequence listed in Table 1 (i.e., any one of SEQ ID NO: 1-276).

[0165] In some embodiments, if the first strand (antisense strand) of the nucleic acid of this application contains a continuous nucleoside sequence of at least 17 nucleosides, then the first strand is related to... SLC25A5 The continuous portions of the mRNA are partially complementary, wherein at least 14, 15, 16, or 17 nucleotides of the continuous nucleotide sequence are complementary to... SLC25A5 The continuous portions of the mRNA are complementary. In some embodiments, the first strand of the nucleic acid of this application comprises a continuous nucleoside sequence of at least 17 nucleotides, wherein at least 14, 15, 16, or 17 nucleotides of the continuous nucleoside sequence are complementary to the continuous portions of any sequence listed in Table 1 (i.e., any one of SEQ ID NO: 1-276). In some embodiments, the first strand of the nucleic acid of this application comprises a continuous nucleoside sequence of 19 nucleotides, wherein at least 14, 15, 16, 17, 18, or all 19 nucleotides of the continuous nucleoside sequence are complementary to the continuous portions of any sequence listed in Table 1 (i.e., any one of SEQ ID NO: 1-276). In some embodiments, the first strand of the nucleic acid of this application comprises a continuous nucleoside sequence of 23 nucleotides, wherein at least 18, 19, 20, 21, 22, or all 23 nucleotides of the continuous nucleoside sequence are complementary to the continuous portions of any sequence listed in Table 1 (i.e., any one of SEQ ID NO: 1-276).

[0166] In some embodiments, the nucleic acid (e.g., siRNA) of this application comprises a sense strand that is substantially or partially complementary to an antisense oligonucleotide, which in turn is complementary to a target gene sequence, and comprises a continuous nucleoside sequence. The nucleoside sequence of the sense strand typically has at least about 80% complementarity with the equivalent region of the nucleoside sequence of the antisense strand over its entire length, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementarity.

[0167] In some embodiments, the nucleic acid (e.g., siRNA) of this application includes an antisense strand that is substantially or partially complementary to the target sequence, and contains a continuous nucleoside sequence that is at least 80% complementary to the target sequence over its entire length, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.

[0168] As used herein, "subject" refers to an animal, such as a mammal, including primates (e.g., humans, non-human primates such as monkeys and chimpanzees), or a non-primate or bird, that expresses the target gene endogenously or heterologously when the target gene sequence has sufficient complementarity with a nucleic acid sequence (e.g., an iRNA agent) to facilitate target knockdown. In some preferred embodiments, the subject is a human.

[0169] The term "treatment" refers to a beneficial or desired outcome, including but not limited to the reduction or improvement of one or more symptoms related to gene expression. "Treatment" can also refer to an extension of life expectancy compared to the expected life expectancy without treatment.

[0170] As used herein, the term “prevent / prevention” is defined as eliminating or reducing the likelihood of the occurrence of one or more symptoms of a disease or condition. For example, the inhibitors disclosed herein may be used to prevent the occurrence of metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or diseases or conditions associated with lipogenesis.

[0171] As used herein, “therapeutic effective amount” is intended to include an amount of nucleic acid (e.g., iRNA) that, when administered to a patient for the treatment of a disease, is sufficient to effectively treat the disease (e.g., by reducing, improving, or maintaining existing disease or symptoms of one or more diseases or their associated comorbidities).

[0172] The phrase “pharmaceutically acceptable” in this document refers to a compound, material, composition, or dosage form that is suitable for contact with tissues in human and animal subjects without excessive toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable benefit / risk ratio.

[0173] As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or solvent, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, for the delivery or transport of a target compound from one organ or site of the body to another. Each carrier must be “acceptable,” meaning it is compatible with other components in the formulation and will not cause harm to the subject receiving treatment.

[0174] When listing the numerical values ​​or ranges of parameters, it means that the values ​​and ranges between the listed values ​​are also part of this application.

[0175] The articles “a” and “a kind” are used in this text to refer to one or more (i.e., at least one) of the grammatical objects of the article.

[0176] The term “including” is used in this document to mean the phrase “including but not limited to”, and may be used interchangeably with it.

[0177] The term “or” is used in this document to mean the term “and / or” and may be used interchangeably with it unless the context clearly indicates otherwise. For example, “justice chain or antisense chain” should be understood as “justice chain or antisense chain or justice chain and antisense chain”.

[0178] The term “about” is used herein to indicate a typical tolerance range in this field. For example, “about” can be understood as approximately 2 standard deviations away from the mean. In some implementations, about means +10%. In some implementations, about means +5%. When “about” appears before a series of numbers or ranges, it should be understood that “about” may modify each number in that series or range.

[0179] The term "at least" preceding a number or series of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as is clearly evident from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in that series or range.

[0180] As used herein, “not more than” or “less than” should be understood as the value adjacent to the phrase and as logically lower or an integer, as logically as the context, up to zero. For example, a double strand with “not more than 2 nucleotides” has 2, 1, or 0 nucleotides. When “not more than” appears before a series of numbers or ranges, it should be understood that “not more than” can modify each number in the series or range.

[0181] The terminal region of the chain refers to the last 5 nucleotides from the 5' or 3' end.

[0182] The nucleobase sequence refers to the sequence of nucleic acid bases in an oligomer.

[0183] Those skilled in the art can combine various embodiments of this application as needed.

[0184] target The inhibitory targets disclosed in this article may be, but are not limited to, mRNA, peptides, proteins, or genes.

[0185] These targets are those that, when inhibited, can help prevent and / or treat metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity and / or diseases or conditions associated with lipogenesis, and / or help reduce lipogenesis.

[0186] The target of repression is the gene SLC25A5 or its gene product, such as mRNA transcribed from the SLC25A5 gene or the ANT2 protein, and repression can be achieved by inhibiting the expression or function of the SLC25A5 / ANT2 gene or protein or both.

[0187] In a preferred embodiment, the target is mRNA expressed from the SLC25A5 gene. Exemplary target sequences on SLC25A5 mRNA are listed in Table 1 below.

[0188] Table 1 below provides the oligonucleotide mRNA target sequence of SLC25A5 and the corresponding position in transcript ENST00000317881.9. It should be understood that SEQ ID NO: 1 to 276 refer to human (Homo sapiens) mRNA sequences.

[0189] Table 1

[0190] It should be understood that SEQ ID NOs: 1 to 276 involve human (Homo sapiens) mRNA sequences.

[0191] Disease / condition This application also provides treatment methods for subjects in need of this treatment. The treatment methods of this application include administering a therapeutically effective amount of the nucleic acid (e.g., siRNA) of this application, such as a nucleic acid targeting SLC25A5 (e.g., siRNA), or a pharmaceutical composition containing a nucleic acid targeting SLC25A5, to a subject (e.g., a subject who could benefit from reduced or suppressed expression of the SLC25A5 gene). The disease to be treated is related to a metabolic disorder or condition, such as a metabolic disorder or condition associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or a disease or condition related to lipogenesis.

[0192] The disease to be treated is associated with a metabolic disorder or condition, such as a metabolic disorder or condition associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or a disease or condition associated with lipogenesis.

[0193] The inhibitors of this application may be used for the prevention and / or treatment of metabolic diseases or conditions. As used herein, the term "metabolic disease" refers to a disease or condition that affects the metabolic processes of a subject and is generally caused by a disorder of normal metabolism.

[0194] The patients to be treated may be those who already have a metabolic disease or condition, or those at risk of developing a metabolic disease or condition. That is, in some embodiments, the inhibitors of this application can be used to treat and / or control existing metabolic diseases or conditions. Treating and / or controlling existing metabolic diseases or conditions with the inhibitors of this application can prevent the worsening of the metabolic disease or condition and / or reverse it. In some cases, treating existing metabolic diseases or conditions with the inhibitors of this application can even cure them. In some embodiments, the inhibitors of this application can be used to prevent the onset of metabolic diseases or conditions in patients at risk of developing them.

[0195] Those skilled in the art can diagnose whether a patient has a metabolic disease or condition, or is at risk of developing one. For example, a metabolic disease or condition can be diagnosed based on weight gain and / or one or more blood markers (including, but not limited to, blood glucose levels, blood insulin levels, blood free fatty acid levels, blood glycated hemoglobin (HbA1c) levels, blood fibrinogen levels, blood cholesterol levels, and blood triglyceride levels). Those skilled in the art understand thresholds for one or more blood markers that indicate the presence of a metabolic disease or condition, or the risk of developing one.

[0196] In some implementations, the metabolic disease is fatty liver, particularly non-alcoholic fatty liver disease (NAFLD). As used herein, “fatty liver” refers to a condition in which excessive fat accumulates in the liver and can lead to serious illnesses such as chronic hepatitis and cirrhosis. In patients with fatty liver, lipids, especially triglycerides, accumulate in hepatocytes to an amount exceeding physiological limits. Biochemically, the criterion for diagnosing fatty liver is that the weight of triglycerides is approximately 10% (100 mg / g wet weight) or more of the liver tissue. Fatty liver is usually detected by observing elevated levels of liver-specific enzymes in the serum, such as transaminases ALT and AST, which are indicators of hepatocyte damage. It can also be detected by the presence of symptoms, including fatigue and pain in the liver area, but a definitive diagnosis usually requires a biopsy and can be supported by imaging studies such as ultrasound and / or MRI. As used herein, the term “NAFLD” or “non-alcoholic fatty liver disease” refers to a disease caused by fat deposition in the liver (steatodegenesis) rather than excessive alcohol consumption. It is associated with insulin resistance and metabolic syndrome.

[0197] In a preferred embodiment, fatty liver is non-alcoholic steatohepatitis (NASH). As used herein, NASH refers to a liver disease characterized by the accumulation of fat (lipid droplets) and inflammation and degeneration of hepatocytes. Once it develops, the disease is associated with a high risk of cirrhosis, a state of altered liver function that can progress to liver failure. Subsequently, NASH often progresses to liver cancer.

[0198] In some embodiments, this application relates to the use of an inhibitor according to this application for reducing one or more of steatosis, lobular inflammation, and / or hepatocellular ballooning degeneration.

[0199] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of diseases or conditions associated with increased steatosis, increased lobular inflammation, and / or increased hepatocellular ballooning degeneration.

[0200] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of fatty liver (e.g., NAFLD or NASH), wherein the inhibitor of this application results in one or more of the following: reduction of steatosis, reduction of lobular inflammation, and / or reduction of hepatocellular ballooning degeneration.

[0201] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of hepatic steatosis.

[0202] In some embodiments, this application relates to the use of the inhibitor according to this application for the treatment and / or prevention of lobular inflammation of the liver.

[0203] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of hepatocellular ballooning degeneration.

[0204] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment or prevention of liver fibrosis in a patient.

[0205] In some embodiments, this application relates to an inhibitor according to this application for the treatment or prevention of diseases or conditions associated with liver fibrosis.

[0206] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of fatty liver (e.g., NAFLD or NASH), wherein the inhibitor of this application reduces the fibrosis stage.

[0207] Those skilled in the art are aware of methods for determining the staging of hepatic steatosis, lobular inflammation, hepatocellular ballooning degeneration, or fibrosis in patients.

[0208] In some embodiments, this application relates to the use of an inhibitor according to this application for reducing one or more of the following levels in a patient: alanine aminotransferase (ALT), aspartate aminotransferase (AST), tissue inhibitor of metalloproteinases-1 (TIMP-1), and / or type III procollagen peptide (PIIINP).

[0209] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of diseases associated with elevated levels of one or more of the following in a patient: alanine aminotransferase (ALT), aspartate aminotransferase (AST), tissue inhibitor of metalloproteinases-1 (TIMP-1), and / or type III procollagen peptide (PIIINP).

[0210] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of metabolic diseases or conditions associated with elevated levels of one or more of the following in a patient: alanine aminotransferase (ALT), aspartate aminotransferase (AST), tissue inhibitor of metalloproteinases-1 (TIMP-1), and / or type III procollagen peptide (PIIINP).

[0211] In some embodiments, this application relates to an inhibitor according to this application for the treatment and / or prevention of metabolic diseases or conditions, wherein the inhibitor of this application reduces one or more of the following levels in a patient: alanine aminotransferase (ALT), aspartate aminotransferase (AST), tissue inhibitor of metalloproteinases-1 (TIMP-1), and / or type III procollagen peptide (PIIINP).

[0212] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of fatty liver (e.g., NAFLD or NASH), wherein the inhibitor of this application reduces one or more of the following levels in a patient: alanine aminotransferase (ALT), aspartate aminotransferase (AST), tissue inhibitor of metalloproteinases-1 (TIMP-1), and / or type III procollagen peptide (PIIINP).

[0213] Those skilled in the art are familiar with methods and commercially available kits for measuring ALT, AST, TIMP-1, and PIIINP levels in patients.

[0214] In some embodiments, this application relates to the use of the inhibitor according to this application for reducing the liver weight to body weight ratio.

[0215] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of diseases or conditions associated with increased liver weight.

[0216] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of metabolic diseases or conditions associated with increased liver weight.

[0217] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of metabolic diseases or conditions, wherein the inhibitor of this application reduces the ratio of liver weight to body weight in a patient.

[0218] In some embodiments, this application relates to the use of an inhibitor according to this application for the treatment and / or prevention of fatty liver (e.g., NAFLD or NASH), wherein the inhibitor of this application reduces the ratio of liver weight to body weight in patients.

[0219] In some implementations, the metabolic disease is obesity. As used herein, the term “obesity” refers to a state in which the natural energy reserves stored in the adipose tissue of an animal (particularly humans and other mammals) increase to a level associated with certain health conditions or increased mortality. As used herein, the term “obesity,” for adults, is defined as a body mass index (BMI) greater than 30. Obesity is generally associated with excessive weight gain, particularly diet-induced weight gain. “(Diet-induced) weight gain” is defined herein as weight gain resulting from excessive dietary intake, including excessive dietary intake of fat, particularly saturated fat, and optionally excessive dietary intake of monosaccharides, including sucrose and fructose. For a given subject, excessive dietary intake (particularly fat and optionally monosaccharides) refers to the intake of dietary amounts (particularly fat and optionally monosaccharides) exceeding the amount required to meet physiological needs and maintain the subject’s energy balance. The effect of treatment on reducing or preventing diet-induced weight gain in a subject can be assessed by comparing the weight gain observed in a treated subject with the weight gain observed in the same subject who did not receive the same diet and had the same level of physical activity.

[0220] In some implementations, a patient is considered at risk of obesity if their BMI is greater than 25. In other implementations, a patient is considered obese if their BMI is greater than 30.

[0221] As used in this article, the term "body mass index" refers to the ratio of weight (in kilograms) to the square of height (in meters).

[0222] "Diseases related to lipogenesis" refer to medical conditions characterized by the abnormal proliferation and differentiation of adipocytes / fat cells, leading to excessive accumulation of adipose tissue. These conditions often result in health complications such as obesity, metabolic disorders, and related comorbidities.

[0223] In this application, "reducing lipogenesis" refers to a medical or pharmaceutical intervention aimed at reducing the formation and accumulation of fat in the body. The reduction in lipogenesis can be determined and / or quantified based on the size and / or number of adipocytes in tissue samples obtained from a patient. Alternatively, the reduction in lipogenesis can be determined and / or quantified by gene expression analysis, detection of lipogenesis markers (i.e., by ELISA), assessment of lipid accumulation in the sample, and / or measurement of triglyceride levels in cells or tissues.

[0224] Therefore, in one specific embodiment, this application relates to an inhibitor suitable for or used to prevent and / or treat metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or diseases or conditions associated with lipogenesis, and / or an inhibitor for reducing lipogenesis.

[0225] Inhibitors The inhibitors of this application include nucleic acids (e.g., siRNA), antibodies, and their antigen-binding fragments (e.g., monoclonal antibodies, peptides, antibody-drug conjugates, and small molecules). Nucleic acids, such as siRNA, are preferred.

[0226] The preferred features of the inhibitors (e.g., oligonucleotides of siRNA) of this application are as follows.

[0227] In some embodiments, the nucleic acid comprises a first strand containing a sequence at least partially complementary to a portion of the RNA transcribed from the SLC25A5 gene (SEQ ID NO: 1381). In a preferred embodiment, the nucleic acid comprises a first strand containing a sequence at least partially complementary to the SLC25A5 mRNA.

[0228] In some implementations, it is used to suppress SLC25A5 The nucleic acid expressing the gene comprises a double-stranded segment, the double-stranded segment comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) with transcription SLC25A5 A portion of the gene's RNA is at least partially complementary, and (ii) Contains at least 17 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 277-552 by 0 or 1.

[0229] In some embodiments, the first chain contains nucleosides 2-18 of any of the sequences shown in SEQ ID NO: 277-552.

[0230] In some implementations, the first chain contains any one of SEQ ID NO: 277-552.

[0231] In some embodiments, the second chain comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 553-828 by 0 or 1, wherein the second chain has a region that is at least 85% complementary to the first chain on the 17 consecutive nucleosides.

[0232] In some implementations, the second chain comprises any one of SEQ ID NO: 553-828.

[0233] In some implementations, it is used to suppress SLC25A5 The nucleic acid expressing the gene comprises a double-stranded segment, which includes a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) with transcription SLC25A5 A portion of the gene's RNA is at least partially complementary, and (ii) Contains at least 21 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 277-552 by 0 or 1.

[0234] In some embodiments, the first chain contains nucleosides 2-22 in any of the sequences shown in SEQ ID NO: 277-552.

[0235] In some embodiments, the second chain comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 553-828 by 0 or 1; wherein the second chain has a region that is at least 85% complementary to the first chain on the 17 consecutive nucleosides.

[0236] In some implementations, the second chain comprises any one of SEQ ID NO: 553-828.

[0237] In some embodiments, the second chain comprises a nucleoside sequence of at least 19 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 553-828 by 0 or 1; wherein the second chain has at least 85% complementary regions to the first chain on the 19 consecutive nucleosides.

[0238] In some embodiments, the second chain comprises a nucleoside sequence of at least 21 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 553-828 by 0 or 1; wherein the second chain has at least 85% complementary regions to the first chain on the 21 consecutive nucleosides.

[0239] In some embodiments, the nucleic acid comprises a first strand comprising a nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 277-552 by 0 or 1, is composed of a nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 277-552 by 0 or 1, or is substantially composed of a nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 277-552 by 0 or 1; And a second chain comprising a nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 553-828 by 0 or 1, consisting of a nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 553-828 by 0 or 1, or consisting substantially of a nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 553-828 by 0 or 1.

[0240] This paper preferably forms a double-chain segment between the first (antonym) chain and the complementary second (just) chain. Table 2 below lists exemplary pairs of complementary antonym chains and just chains.

[0241] Table 2 provides the unmodified first (antisense) strand sequence and the corresponding unmodified second (sense) strand sequence of the siRNA oligonucleotide according to this application, as well as their corresponding positions in the overall gene sequence of SEQ ID NO:1381, as shown below.

[0242] Table 2:

[0243] In one specific embodiment, this application relates to nucleic acids comprising a first strand and a second strand, wherein the first and second strands comprise nucleoside sequences differing from any one of the following first and second sequences by 0 or 1 nucleoside, nucleoside sequences consisting of any one of the following first and second sequences differing by 0 or 1 nucleoside, or nucleoside sequences substantially consisting of any one of the following first and second sequences differing by 0 or 1 nucleoside:

[0244] In a particularly preferred embodiment, this application relates to a nucleic acid comprising a first strand and a second strand, wherein the first and second strands comprise a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, consist of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, or consist substantially of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside:

[0245] In a particularly preferred embodiment, this application relates to nucleic acids comprising a first strand and a second strand, said first and second strands comprising a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside:

[0246] In some implementations, it is used to suppress SLC25A5 The nucleic acid expressing the gene comprises a double-stranded segment, the double-stranded segment comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) with transcription SLC25A5 A portion of the gene's RNA is at least partially complementary, and (ii) Containing at least 17 consecutive nucleosides that differ from any one of SEQ ID NO: 829-1104 by 0 or 1 nucleoside.

[0247] In some embodiments, the first chain contains nucleosides 2-18 of any of the sequences shown in SEQ ID NO: 829-1104.

[0248] In some implementations, it is used to suppress SLC25A5 The nucleic acid expressing the gene comprises a double-stranded segment, the double-stranded segment comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) with transcription SLC25A5 A portion of the gene's RNA is at least partially complementary, and (ii) Contains at least 21 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 829-1104 by 0 or 1.

[0249] In some embodiments, the first chain contains nucleosides 2-22 in any of the sequences shown in SEQ ID NO: 829-1104.

[0250] In some implementations, the first chain contains any one of SEQ ID NO: 829-1104.

[0251] The modification patterns of the nucleic acids shown in SEQ ID NO: 829-1104 are summarized in Table 3 below: Table 3 provides the first (antisense) sequence of the modified siRNA oligonucleotide of this application, and the corresponding unmodified first (antisense) sequence, as shown below.

[0252] Table 3

[0253] In some embodiments, the second chain comprises a nucleoside sequence of at least 17 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 1105-1380 by 0 or 1; wherein the second chain has a region that is at least 85% complementary to the first chain on the 17 consecutive nucleosides.

[0254] In some embodiments, the second chain comprises a nucleoside sequence of at least 19 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 1105-1380 by 0 or 1; wherein the second chain has a region that is at least 85% complementary to the first chain on the 19 consecutive nucleosides.

[0255] In some embodiments, the second chain comprises a nucleoside sequence of at least 21 consecutive nucleosides that differ from any one of the nucleosides in SEQ ID NO: 1105-1380 by 0 or 1; wherein the second chain has a region that is at least 85% complementary to the first chain on the 21 consecutive nucleosides.

[0256] In some implementations, the second chain comprises any one of SEQ ID NO: 1105-1380.

[0257] The modification patterns of the nucleic acids shown in SEQ ID NO: 1105-1380 are summarized in Table 4 below.

[0258] Table 4 provides the modified second (positive) sequence of the siRNA oligonucleotide of this application, as well as the corresponding unmodified second (positive) sequence, as described below.

[0259] Table 4

[0260] As used herein, particularly in Tables 3 and 4, the following abbreviations are used to modify nucleosides: A-adenosine; C-cytidine; G-guanosine; T-thymidine; m-2'-O-methyl; f-2'-fluorine; s-thiophosphate bond; o-thermally unstable nucleoside; ia-reverse abase-free nucleoside.

[0261] Am represents 2'-O-methyladenosine, Cm represents 2'-O-methylcytidine, Gm represents 2'-O-methylguanosine, Um represents 2'-O-methyluridine, Af represents 2'-fluoroadenosine, Cf represents 2'-fluorocytidine, Gf represents 2'-fluoroguanosine, and Uf represents 2'-fluorouridine.

[0262] Furthermore, the letter "s" is used as an abbreviation for the thiophosphate bond between two consecutive (modified) nucleosides. For example, the abbreviation "AmsAm" is used to represent two consecutive 2'-O-methyladenosine nucleosides linked by a 3'5' thiophosphate bond. No abbreviation is used for nucleosides linked by a standard 3'5' phosphodiester bond. For example, the abbreviation "AmAm" is used to represent two consecutive 2'-O-methyladenosine nucleosides linked by a 3'5' phosphodiester bond.

[0263] As shown in Table 4 above, some modified second-strand sequences contain the preferred 5'iaia motif. However, it should also be understood that the range of these modified second-strand sequences also includes Me / F modified second strands without the 5'iaia motif.

[0264] In some embodiments, the nucleic acid comprises a first strand comprising a (modified) nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 829-1104 by 0 or 1 nucleoside, and is composed of or substantially composed of a (modified) nucleoside sequence that differs from any one of the nucleosides in SEQ ID NO: 829-1104 by 0 or 1 nucleoside. And a second chain comprising (modified) nucleoside sequences that differ from any one of the nucleosides in SEQ ID NO:1105-1380 by 0 or 1, or consisting of (modified) nucleoside sequences that differ from any one of the nucleosides in SEQ ID NO:1105-1380 by 0 or 1.

[0265] Preferred combinations of complementary modified antonymous (first) chains and just (second) chains are listed in Table 5 below.

[0266] Table 5 lists the bichains, with the bichain ID referencing the antisense chain ID and justice chain ID modified in the previous Tables 3 and 4.

[0267] Table 5

[0268] For the double strands in Table 5: ETX-M00001351 to ETX-M00001626 preferably have Figure 8b The double-chain structure.

[0269] In a particularly preferred embodiment, this application relates to nucleic acids comprising a first strand and a second strand, the first and second strands comprising a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside:

[0270] In even more preferred embodiments, this application relates to nucleic acids comprising a first strand and a second strand, the first and second strands comprising a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any one of the following first and second sequences by 0 or 1 nucleoside:

[0271] If there is any ambiguity between the sequences in this specification and the sequences in the attached sequence list, the sequences provided herein shall be considered correct sequences.

[0272] non-base nucleotides In some embodiments, the nucleic acid of this application contains one, for example two, for example three, for example four or more abasic nucleosides. Abasic nucleosides are modified nucleosides because they lack a base that normally appears at position 1 of the sugar moiety. Typically, the abasic nucleosides in the nucleic acid of this application have a hydrogen atom at position 1 of the sugar moiety.

[0273] The abasic nucleoside is located in the terminal region of the second chain, preferably within the five terminal nucleosides at the end of the chain. The terminal region can be the five terminal nucleosides, which include the abasic nucleoside.

[0274] The second chain may include the following preferred features (combinations are explicitly considered unless they are mutually exclusive): Two or more abase-free nucleotides in the terminal region of the second chain; and / or Two or more abase-free nucleotides in the 5' or 3' terminal region of the second strand; and / or Two or more abase-free nucleotides in the 5' or 3' terminal region of the second strand, wherein the abase-free nucleotides are located in the overhang described herein; and / or Two or more consecutive abasic nucleosides in the terminal region of the second chain, wherein preferably one of such abasic nucleosides is a terminal nucleoside; and / or Two or more consecutive abasic nucleotides in the 5' or 3' terminal region of the second strand, wherein preferably one such abasic nucleotide is a terminal nucleotide in the 5' or 3' terminal region of the second strand; and / or At least one base-free nucleotide in the terminal region of the second chain is attached to the reverse internucleotide bond of an adjacent basic nucleotide; and / or At least one base-free nucleotide in the 5' or 3' terminal region of the second chain is attached to the reverse nucleotide bond of an adjacent basic nucleotide; and / or The penultimate nucleoside is a baseless nucleoside that is linked via a reverse bond to a non-terminal nucleoside (referred to herein as the penultimate nucleoside); and / or When the strand is read toward the end containing the terminal nucleoside, the baseless nucleoside is connected by a 5'-3' bond as two terminal nucleosides; When the chain is read toward the end containing the terminal nucleoside, the baseless nucleoside is connected by 3'-5' bonds as two terminal nucleosides; As a nucleoside with no bases at the two ends, the penultimate nucleoside is linked to the penultimate nucleoside by a reverse bond, and the reverse bond is either a 5-5' reverse bond or a 3'-3' reverse bond; As a nucleoside without bases at the two terminal positions, the penultimate nucleoside is linked to the penultimate nucleoside via a reverse bond, and in which (1) When read toward the end containing the terminal and the penultimate anucleotide, the reverse bond is a 5-5' reverse bond, and the bond between the terminal and the penultimate anucleotide is a 3'5' bond; or (2) When read toward the end containing the terminal and the penultimate abase-free nucleoside, the reverse bond is a 3-3' reverse bond, and the bond between the terminal and the penultimate abase-free nucleoside is a 5'3'.

[0275] Preferably, the second chain has no nucleoside at its end.

[0276] Preferably, the terminal region of the second chain (preferably at the terminal and penultimate positions) contains two or at least two abase-free nucleosides.

[0277] Preferably, the two or more abasic nucleosides are consecutive; for example, all abasic nucleosides can be consecutive. For example, terminal 1, terminal 2, terminal 3, or terminal 4 nucleotides can be abasic nucleosides.

[0278] Abase-free nucleosides can also be linked to adjacent nucleosides via 5'-3' phosphodiester bonds or reverse bonds, unless there is only one abase-free nucleoside at the end, in which case it will be linked to the adjacent nucleoside via a reverse bond.

[0279] Reverse bonds (also known as inverted linkages, which are common in the art) include 5'-5', 3'-3', 3'-2', or 2'-3' phosphodiester bonds between adjacent sugar moieties of a nucleoside.

[0280] Non-terminal abasic nucleosides have two phosphodiester bonds, one for each adjacent nucleoside. These bonds can be anti-bonds, 5'-3 phosphodiester bonds, or one of these.

[0281] A preferred embodiment includes two abase-free nucleosides at the end and penultimate position of the second chain, wherein the reverse nucleoside bond is located between the penultimate (abase-free) nucleoside and the penultimate nucleoside.

[0282] Preferably, there are two base-free nucleosides at the end and penultimate position of the second chain, and the penultimate nucleoside is linked to the penultimate nucleoside through an inverse nucleoside internucleotide bond and linked to the terminal nucleoside through a 5'-3' or 3'-5' phosphodiester bond (read along the direction of the molecule end).

[0283] Preferably, the nucleic acid according to this application comprises one or more abasic nucleosides, optionally wherein the one or more abasic nucleosides are located in the terminal region of the second chain, and / or wherein at least one abasic nucleoside is linked to an adjacent basic nucleoside via a reverse internucleotide bond.

[0284] The different preferred features are as follows: The reverse nucleoside internucleotide bond is a 3'-3' reverse bond. The reverse nucleoside internucleotide bond is located in the terminal region of the 5' end of the phosphate ester in the second chain.

[0285] The reverse nucleoside internucleotide bond is a 5'-5' reverse bond. The reverse nucleoside internucleotide bond is located in the terminal region distal to the 3' end hydroxyl group of the second chain.

[0286] In some embodiments, the second chain comprises two consecutive abasic nucleosides in its 5' terminal region, one of which is the terminal nucleoside of the 5' terminal region of the second chain, and the other is the penultimate nucleoside of the 5' terminal region of the second chain, wherein: (a) the penultimate abasic nucleoside is connected to an adjacent first basic nucleoside in the adjacent 5' proximal terminal region via a reverse internucleotide bond; (b) the reverse bond is a 5-5' reverse bond; and (c) when read toward the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 3'5'. More typically, (i) the first and second chains each have a length of 23 nucleotides; (ii) two thiophosphate nucleotide bonds are located between three consecutive positions in the 5' proximal region of the second chain, wherein the first thiophosphate nucleotide bond is located between the adjacent first basic nucleotide of (a) and the adjacent second basic nucleotide in the 5' proximal region of the second chain, and the second thiophosphate nucleotide bond is located between the adjacent second basic nucleotide and the adjacent third basic nucleotide in the 5' proximal region of the second chain; (iii) two thiophosphate nucleotide bonds are located between three consecutive positions in the 5' and 3' terminal regions of the first chain, whereby the terminal nucleotides at the 5' and 3' terminal regions of the first chain are each connected to the corresponding 5' and 3' adjacent penultimate nucleotides via thiophosphate nucleotide bonds, and each first 5' and 3' penultimate nucleotide is connected to the corresponding 5' and 3' adjacent penultimate nucleotide via thiophosphate nucleotide bonds; and (iv) The second strand of a nucleic acid is directly or indirectly coupled to one or more ligand moieties in its 3' terminal region.

[0287] Alternatively, the second chain comprises two consecutive abasic nucleosides, preferably at the protruding end of the 3' terminal region of the second chain, one of which is the terminal nucleoside of the 3' terminal region of the second chain, and the other is the penultimate abasic nucleoside of the 3' terminal region of the second chain, wherein: (a) the penultimate abasic nucleoside is connected to the first adjacent abasic nucleoside in the adjacent 3' proximal terminal region by a reverse internucleotide bond; (b) the reverse bond is a 3-3' reverse bond; and (c) when read toward the end containing the terminal and penultimate abasic nucleosides, the bond between the terminal and penultimate abasic nucleosides is 5'-3'. More typically, (i) the first and second chains each have a length of 23 nucleotides; (ii) two thiophosphate nucleoside bonds are respectively between three consecutive positions in the 3' proximal region of the second chain, wherein the first thiophosphate nucleoside bond is between the adjacent first basic nucleoside of (a) and the adjacent second basic nucleoside in the 3' proximal region of the second chain, and the second thiophosphate nucleoside bond is between the adjacent second basic nucleoside and the adjacent third basic nucleoside in the 3' proximal region of the second chain; (iii) two thiophosphate nucleoside bonds are respectively between three consecutive positions in the 5' and 3' terminal regions of the first chain, whereby each terminal nucleoside in the 5' and 3' terminal regions of the first chain is respectively connected to the corresponding 5' and 3' adjacent penultimate nucleoside via a thiophosphate nucleoside bond, and each first 5' and 3' penultimate nucleoside is connected to the corresponding 5' and 3' adjacent penultimate nucleoside via a thiophosphate nucleoside bond; and (iv) The second strand of a nucleic acid is directly or indirectly coupled to one or more ligand moieties in its 5' terminal region.

[0288] Examples of the structures are as follows (where the specific RNA nucleosides shown are non-restrictive and can be any RNA nucleosides): A 3'-3' reverse bond (and also shows the 5'-3 direction of the last phosphodiester bond between the two baseless molecules, read towards the end of the molecule). Example B illustrates the 5'-5' reverse bond (and also shows the 3'-5' orientation of the last phosphodiester bond between two baseless molecules, read towards the end of the molecule). When one or more reverse nucleoside bonds (i.e., 5'-5' or 3'-3' reverse nucleoside bonds) are present, the nucleic acid contains one or more abasic nucleosides. The reverse bond arises from a change in the orientation of adjacent nucleoside sugars, resulting in a 3'-5' orientation rather than the conventional 5'-3' orientation (refer to the numbering of ring atoms on the nucleoside sugar). The one or more abasic nucleosides present in the nucleic acid of this application preferably comprise such reverse nucleoside sugars.

[0289] If the terminal nucleoside has a reverse orientation, it results in an "inverted" terminal configuration for the entire nucleic acid. While some structures drawn and cited in this paper are represented using the conventional 5'-3' orientation (refer to the numbering of ring atoms on the nucleoside sugar), it is understood that the presence of a terminal nucleoside with an orientation change and a proximal 3'-3' reverse bond results in an overall 5'-5' terminal structure for the nucleic acid (i.e., a conventional 3' terminal nucleoside becomes a 5' terminal nucleoside). Alternatively, it can be understood that the presence of a terminal nucleoside with an orientation change and a proximal 5'-5' reverse bond results in an inverted nucleic acid with an overall 3'-3' terminal structure.

[0290] The proximal 3'-3' or 5'-5' reverse bonds described herein may comprise a reverse bond directly adjacent to / connected to a terminal nucleoside with a reverse orientation (e.g., a single terminal nucleoside with a reverse orientation). Alternatively, the proximal 3'-3' or 5'-5' reverse bonds described herein may comprise a reverse bond adjacent to two or more nucleosides with reverse orientation (e.g., two or more terminal region nucleosides with reverse orientation, such as a terminal and penultimate nucleoside). In this way, the reverse bond can connect to the penultimate nucleoside with a reverse orientation. While those skilled in the art will understand that the reverse orientation described above can result in a nucleic acid molecule having the overall 3'-3' or 5'-5' end structure described herein, it should also be understood that if one or more additional reverse bonds and / or nucleosides with reverse orientation are present, the entire nucleic acid may have a 3'-5' end structure relative to the conventional 5' / 3' end.

[0291] In one respect, nucleic acids can have 3'-3' reverse bonds, and the terminal sugar moiety can contain a 5' hydroxyl group instead of a 5' phosphate group at the 5' position of the terminal sugar.

[0292] Therefore, those skilled in the art will clearly understand that the 5'-5', 3'-3', and 3'-5' (read along the terminal direction) terminal variants of the more conventional 5'-3' structure (refer to the ring atom numbering on the terminal nucleotide sugar) drawn herein are all included within the scope of this disclosure, wherein one or more reverse bonds are present.

[0293] For example, where a reverse nucleoside bond and / or one or more nucleosides with reverse orientations produce a reverse end, and the relative position of the bond (e.g., with a linker) or the position of an internal feature (e.g., a modified nucleoside) is defined relative to the 5' or 3' end of the nucleic acid, then the 5' or 3' end is the conventional 5' or 3' end that would exist without the reverse bond, wherein the conventional 5' or 3' end is determined by taking into account the directionality and / or nucleoside orientation of most internal nucleoside bonds within the nucleic acid. From these internal bonds and / or nucleoside orientations, it can be determined which ends of the nucleic acid would constitute the conventional 5' and 3' ends of the molecule in the absence of a reverse bond (refer to the ring atom numbering on the terminal nucleoside sugar).

[0294] For example, in the structure shown below, the first two positions at the "5'" end have no base residues. If the terminal nucleotide has a reverse orientation, the "5'" end shown in the diagram below (i.e., the conventional 5' end) might actually contain a 3' OH, given the reverse nucleotide at the terminal position. However, when read according to the standard 5' [PO4] to 3' [OH] orientation of nucleic acid molecules (refer to the ring atom numbering on the nucleotide sugar), the majority of the molecule will contain a conventional nucleotide internucleotide bond from the 3' OH of the sugar to the 5' phosphate of the next sugar. This can be used to determine the conventional 5' and 3' ends in the absence of a reverse terminal configuration.

[0295] A 5' AA-Me-Me-Me-Me-Me-Me-F-Me-FFF-Me-Me-Me-Me-Me-Me-Me-Me-Me-Me3' The reverse bond is preferably located at the end of the nucleic acid (e.g., RNA), at the distal end of the ligand portion of the molecule (e.g., the portion containing GalNAc).

[0296] GalNAc-siRNA constructs with 5'-GalNAc on the positive strand can have a reverse bond at the other end of the positive strand.

[0297] GalNAc-siRNA constructs with 3'-GalNAc on the positive strand can have a reverse bond at the other end of the positive strand.

[0298] In some embodiments, the second (positive) strand of the nucleic acid in this application contains two consecutive anucleotides at the 5' end region, as shown in the following 5' end motif. in: B represents a nucleoside base. T represents H, OH, or 2' ribose modification. Z represents the remaining nucleosides in the second chain.

[0299] In some embodiments, the second strand (sense strand) of the nucleic acid of this application contains two consecutive anucleotides at the 5' end region, as shown in the following 5' end motif. in: B represents a nucleoside base. T represents H, OH, or 2' ribose modification (preferably 2' ribose modification, more preferably 2' Me or 2' F ribose modification). V represents O or S (O is preferred). R represents H or C 1-4 Alkyl (preferably H), Z represents the remaining nucleoside in the second chain. More preferably, the following 5' terminal motifs in: B represents a nucleoside base. T represents 2' ribose modification (preferably 2'Me or 2'F ribose modification), Z represents the remaining nucleosides in the second chain.

[0300] The reverse bond is preferably located at the end of the nucleic acid (e.g., RNA), which is distal to the ligand portion of the molecule (e.g., the portion containing GalNAc).

[0301] GalNAc-siRNA constructs with 5'-GalNAc on the positive strand can have a reverse bond at the other end of the positive strand.

[0302] GalNAc-siRNA constructs with 3'-GalNAc on the positive strand can have a reverse bond at the other end of the positive strand.

[0303] In a preferred embodiment, the second (positive) strand of the nucleic acid of this application contains two consecutive anucleotides in the 5' end region, as shown in the following 5' end motif.

[0304] in: B represents a nucleoside base. T represents H, OH, or 2' ribose modification (preferably 2' ribose modification, more preferably 2' Me or 2' F ribose modification). V represents O or S (O is preferred). R represents H or C 1-4 Alkyl (preferably H), Z contains 11 to 26 consecutive nucleotides, preferably 15 to 21 consecutive nucleotides, more preferably 19 consecutive nucleotides, and more preferably the following 5' terminal motif. in: B represents a nucleoside base. T represents 2' ribose modification (preferably 2'Me or 2'F ribose modification), Z contains 19 consecutive nucleosides.

[0305] Nucleic acid length In one aspect, i) the length of the first nucleic acid chain is in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides; and / or ii) the length of the second nucleic acid chain is in the range of 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 21 nucleosides.

[0306] Typically, the length of the double-stranded nucleic acid segment is between 17 and 30 nucleotides, more preferably 19 or 21 nucleotides. Similarly, the length of the complementary region between the first strand and a portion of the RNA transcribed from the SLC25A5 gene is between 17 and 30 nucleotides.

[0307] Typically, the double-stranded structure of nucleic acids (such as iRNA) is approximately 15 to 30 base pairs in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 1 9-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths between the above ranges and lengths are also considered part of this application.

[0308] Similarly, the length of the complementary regions between the antisense and target sequences and / or between the antisense and sense sequences is approximately 15 to 30 nucleotides, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21. 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleosides. Ranges and lengths between the above ranges and lengths are also considered part of this application.

[0309] In some preferred embodiments, the length of the complementary region between the antisense sequence and the target sequence and / or the complementary region between the antisense sequence and the sense sequence is at least 17 nucleotides. For example, the length of the complementary region between the antisense strand and the target is 19 to 21 nucleotides, for example, the length of the complementary region is 21 nucleotides.

[0310] In a preferred embodiment, the length of each chain does not exceed 30 nucleosides.

[0311] In some preferred embodiments, the length of the double-stranded structure of the nucleic acid (e.g., siRNA) is 19 or 21 base pairs. In particularly preferred embodiments, the double-stranded structure may have one of the following structures: For example, ETX-M00001351–ETX-M00001626 or The nucleic acids (e.g., dsRNA) described herein may also include one or more single-stranded nucleoside overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleosides. The nucleoside overhangs may contain or consist of nucleosides / nucleoside analogs (including deoxynucleosides / nucleosides). The overhangs may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleosides of the overhangs may be at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the nucleic acid (e.g., dsRNA).

[0312] In some preferred embodiments, at least one chain contains a 3' overhang of at least one nucleoside, for example, at least one chain contains a 3' overhang of at least two nucleosides. The overhangs are suitably located on the antisense / guide chain and / or the sense / passenger chain.

[0313] Nucleic acid modification In some embodiments, the nucleic acid (e.g., RNA, such as dsiRNA) of this application does not contain further modifications, such as chemical modifications or conjugations known in the art and described herein.

[0314] In other preferred embodiments, the nucleic acid (e.g., RNA, such as dsiRNA) of this application is further chemically modified to enhance stability or other beneficial properties.

[0315] In some embodiments of this application, almost all nucleosides are modified.

[0316] The nucleic acids described in this application can be synthesized or modified using methods well established in the art, such as those described in "Currentprotocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), JohnWiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference.

[0317] Modifications include, for example, end modifications, such as 5' end modifications (phosphorylation, coupling, reverse bond) or 3' end modifications (coupling, DNA nucleoside within RNA or RNA nucleoside within DNA, reverse bond, etc.); base modifications, such as substitution with a stable base, an unstable base, or a base that pairs with an expanded library of paired bases, or a conjugated base; sugar modifications (e.g., at the 2'-position or 4'-position) or sugar substitution; or backbone modifications, including modifications or substitutions of phosphodiester bonds.

[0318] Specific examples of nucleic acid (e.g., siRNA) compounds that can be used in the embodiments described herein include, but are not limited to, RNA containing a modified backbone or lacking natural nucleoside internucleotide bonds. Nucleic acids (e.g., RNA) having a modified backbone include, but are not limited to, nucleic acids whose backbone does not contain phosphorus atoms. For the purposes of this specification, and as sometimes cited in the art, modified nucleic acids (e.g., RNA whose internucleotide backbone does not contain phosphorus atoms) may also be considered oligonucleotides. In some embodiments, modified nucleic acids (e.g., siRNA) contain phosphorus atoms in their internucleotide backbone.

[0319] Modified nucleic acid (e.g., RNA) backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methylphosphonates and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), thioaminophosphates, thioalkylphosphonates, thioalkyl phosphate triesters, and borane phosphates having normal 3'-5' bonds, 2'-5' linked analogs of these compounds, and those with opposite polarities (where adjacent nucleoside unit pairs are linked in 5'-3' or 5'-2'). Various salts, mixed salts, and free acid forms are also included.

[0320] Modified nucleic acids (e.g., RNA) may also contain one or more substituted sugar moieties. The nucleic acids described herein (e.g., siRNA, such as dsiRNA) may include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted. Preferred modifications are 2'-O-methyl and 2'-F.

[0321] In some preferred embodiments, the nucleic acid contains at least one modified nucleoside.

[0322] The nucleic acid of this application may contain one or more modified nucleosides on the first and / or second strand.

[0323] In some implementations, almost all nucleosides of the sense strand and all nucleosides of the antisense strand contain modifications.

[0324] In some implementations, all nucleosides in the sense strand and almost all nucleosides in the antisense strand contain modifications.

[0325] In some implementations, all nucleosides in the sense strand and all nucleosides in the antisense strand contain modifications.

[0326] In one embodiment, at least one of the modified nucleosides is selected from the group consisting of: deoxynucleosides, 3'-terminal deoxythymidine (dT) nucleosides, 2'-O-methyl modified nucleosides (also referred to herein as 2'-Me, where Me is methoxy), 2'-fluorine modified nucleosides, 2'-deoxy modified nucleosides, locked nucleosides, non-locked nucleosides, conformationally restricted nucleosides, restricted ethyl nucleosides, baseless nucleosides, 2'-amino modified nucleosides, 2'-O-allyl modified nucleosides, and 2'-... Nucleosides modified with -C-alkyl groups, 2'-hydroxy groups, 2'-methoxyethyl groups, 2'-O-alkyl groups, morpholino groups, aminophosphates, nucleosides containing non-natural bases, tetrahydropyran-modified nucleosides, 1,5-dehydrohexyl alcohol-modified nucleosides, cyclohexenyl-modified nucleosides, nucleosides containing thiophosphate groups, nucleosides containing methylphosphonic acid groups, nucleosides containing 5'-phosphate groups, and nucleosides containing 5'-phosphate mimics. In another embodiment, the modified nucleoside comprises a short sequence of a 3'-terminal deoxythymidine (dT).

[0327] The modification on the nucleoside may preferably be selected from, but is not limited to, LNA, HNA, CeNA, 2-methoxyethyl, 2'-O-alkyl, 2-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxy, and combinations thereof. In another embodiment, the modification on the nucleoside is a 2-O-methyl (“2-Me”) or 2'-fluoro modification.

[0328] A preferred modification is the modification at the 2'-OH group of the ribose, optionally selected from 2'-Me or 2'-F modifications.

[0329] In some embodiments, the nucleic acid (e.g., an RNAi agent) also contains at least one phosphate thioester or methylphosphonate nucleoside bond. For example, the phosphate thioester or methylphosphonate nucleoside bond may be at the 3'-end or at the end region of one strand (i.e., the sense strand or the antisense strand); or at the ends of both strands (the sense strand and the antisense strand).

[0330] In some embodiments, the thiophosphate or methylphosphonate nucleoside internucleotide bond is at the 5' end or the terminal region of one strand (i.e., the sense strand or the antisense strand); or at the end of both strands (i.e., the sense strand and the antisense strand).

[0331] In some embodiments, the thiophosphate or methylphosphonate nucleoside internucleotide bond is at the 5'-end and 3'-end, or at the end region of one chain (i.e., the sense chain or the antisense chain); or at the end of both chains (i.e., the sense chain and the antisense chain).

[0332] Any nucleic acid may contain one or more phosphate thioester (PS) modifications, such as containing at least two PS nucleoside bonds at the end of the chain.

[0333] At least one of the oligonucleotide chains preferably contains at least two consecutive thiophosphate modifications in the last three nucleotides of the oligonucleotide.

[0334] Therefore, this application also relates to the nucleic acids disclosed herein, which contain thiophosphate nucleoside bonds between at least two or three consecutive positions, for example, in the 5' and / or 3' terminal regions and / or near-terminal regions of the second strand, wherein the near-terminal regions are preferably adjacent to the terminal regions of one or more base-free nucleosides of the second strand.

[0335] This article discloses a nucleic acid that contains phosphate thioester nucleoside bonds between at least two or three consecutive positions in the 5' and / or 3' terminal regions of the first strand, wherein preferably, the terminal positions of the 5' and / or 3' terminal regions of the first strand are connected to their adjacent positions by phosphate thioester nucleoside bonds.

[0336] The nucleic acid chain can be RNA, which contains thiophosphate nucleoside bonds between three adjacent nucleosides of two adjacent terminal abasic nucleosides.

[0337] The preferred nucleic acid is a double-stranded RNA, which contains two adjacent abase-free nucleotides at the 5' end of the second strand and a ligand portion containing one or more GalNAc ligand portions at the 3' end of the second strand. More preferably, the same nucleic acid may also contain phosphate thioester bonds between nucleotides at positions 3-4 and 4-5 of the second strand, read from position 1 of the second strand.

[0338] Position 1 of the first or second strand refers to the nucleoside closest to the end of the nucleic acid (ignoring any abase nucleosides), and this nucleoside is connected to the adjacent nucleoside (at position 2) by internal bonds from 3' to 5', referring to the bonds between the backbone sugar portions, and read in the direction away from the end of the molecule.

[0339] Therefore, it can be seen that "position 1 of the positive strand" refers to the nucleoside closest to the 5' end of the normal positive strand (excluding nucleosides without bases). Typically, the nucleoside at position 1 of the positive strand is equivalent to the 5' nucleoside of the selected target nucleic acid sequence, and more generally, the positive strand starts from position 1 of the positive strand and has nucleosides equivalent to the target nucleic acid sequence, while also allowing for acceptable mismatches between sequences.

[0340] As used in this article, "position 1 of the antisense strand" refers to the nucleoside closest to the 5' end of the conventional antisense strand (excluding nucleosides without bases). As mentioned earlier, there are complementary regions between the sense and antisense strands; therefore, the antisense strand also has regions complementary to the aforementioned target nucleic acid sequence.

[0341] Preferred modifications that can be used with the sequence according to this application may be as follows: Modification 1: First chain modification: NmsNfsNmNfNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNfNfNfNfNfNmNmNmNmNmNmNmNmNfNmNm (5' to 3') Modification 2: First chain modification: NmsNfsNmNfNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNfNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNm (5' to 3') Modification 3: First chain modification: NmsNfsNmNfNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNm (5' to 3') Modification 4: First chain modification: NmsNfsNmNfNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNfNmNfNfNfNfNfNmNmNmNmNmNmNmNmNm (5' to 3') Modification 5: First chain modification: NmsNfsNmNmNmNfNmNmNfNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 6: First chain modification: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNfNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 7: First chain modification: NmsNfsNmNmNmNyNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 8: First chain modification: NmsNfsNmNmNmNyNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 9: First chain modification: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNfNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 10: First chain modification: NmsNfsNmNfNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 11: First chain modification: NmsNfsNmNfNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 12: First chain modification: NmsNfsNmNmNmNfNmNfNfNmNmNmNmNfNmNfNmNfNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') Modification 13: First chain modification: NmsNfsNmNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNfNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') In each of the above modifications: ia represents a reverse abase-free nucleoside; Nm represents a nucleoside modified with 2'Me ribose; Nf represents a nucleoside modified with 2'F ribose; Ny represents a nucleoside with a thermally unstable modification, preferably wherein the unstable modification is selected from modified unlocked nucleic acids (UNA) and ethylene glycol nucleic acids (GNA), more preferably ethylene glycol nucleic acids, and most preferably (S)-ethylene glycol nucleic acids; s represents the internucleotide bond between thiophosphate esters.

[0342] Particularly preferred modifications that can be used with the sequence according to this application may be: Modification 6: First chain modification: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNfNmNmNmsNmsNm (5' to 3') Second chain modification: iaiaNmsNmsNmNmNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm (5' to 3') In each of the above modifications: ia represents a reverse abase-free nucleoside; Nm represents a 2'Me ribose-modified nucleoside; and s represents the internucleotide bond between thiophosphate esters.

[0343] Nucleic acid-ligand coupling Another modification of the nucleic acid (e.g., RNA, such as siRNA) in this application involves linking the nucleic acid (e.g., siRNA) to one or more ligand moieties, for example, to enhance the activity, cellular distribution, or cellular uptake of the nucleic acid (e.g., siRNA), such as entry into the cell.

[0344] In some embodiments, the inhibitor of this application is conjugated to a ligand moiety capable of and / or promoting hepatocyte targeting. In some embodiments, hepatocyte targeting is achieved using an N-acetylgalactosamine (GalNAc) conjugate, as described in more detail below. That is, in some embodiments, the inhibitor of this application is a siRNA-GalNAc conjugate.

[0345] In some embodiments, the ligand moiety can be linked to a nucleic acid (e.g., siRNA oligonucleotide) via a cleavable or non-cleavable linker. The term "linker" or "linking group" refers to the organic part that connects two parts of a compound (e.g., covalently connects two parts of a compound).

[0346] The ligand can attach to the 3' or 5' end of the justice chain.

[0347] The ligand is preferably coupled to the 3' end of the positive strand of a nucleic acid (e.g., siRNA agent).

[0348] Therefore, this application relates in another aspect to conjugates for inhibiting the expression of targets (e.g., target genes) in cells, said conjugates comprising a nucleic acid portion and one or more ligand portions, said nucleic acid portion comprising the nucleic acids disclosed herein.

[0349] In one aspect, the second strand of a nucleic acid is coupled directly or indirectly (e.g., via a linker) to one or more ligand moieties, wherein the ligand moieties are typically in the terminal region of the second strand, preferably in its 3' terminal region.

[0350] In some implementations, the ligand portion comprises GalNAc or a GalNAc derivative that is linked to a nucleic acid (e.g., dsiRNA) via a linker.

[0351] Therefore, this application relates to a coupling compound wherein the ligand portion comprises i) One or more GalNAc ligands; and / or ii) One or more GalNAc ligand derivatives; and / or iii) One or more GalNAc ligands coupled to the nucleic acid via a linker.

[0352] The GalNAc ligand can be directly or indirectly coupled to the 5' or 3' terminal region of the second strand of nucleic acid, preferably coupled to its 3' terminal region.

[0353] GalNAc ligands are well known in the art, especially as described in EP3775207A1.

[0354] In some implementations, the ligand portion comprises one or more ligands.

[0355] In some implementations, the ligand portion comprises one or more carbohydrate ligands.

[0356] In some implementations, one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and / or polysaccharides.

[0357] 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.

[0358] In some implementations, one or more carbohydrates contain one or more N-acetylgalactosamine moieties.

[0359] In some embodiments, the compounds described anywhere herein comprise two or three N-acetylgalactosamine moieties.

[0360] In some implementations, one or more ligands are connected in a linear or branched configuration, for example, each configuration is connected to a branch point of the entire junction.

[0361] Figure 1a and 1b Exemplary linear and branching configurations are shown: exist Figure 1a In (linear), (a) and / or (b) can typically represent linking bonds or groups, such as phosphate esters or thiophosphate ester groups.

[0362] exist Figure 1b In some embodiments, one or more ligands are linked in a biantennary or triantennary branching configuration. Typically, a triantennary branching configuration, such as the N-acetylgalactosamine triantennary branching configuration, is preferred.

[0363] connector Exemplary compounds of this application include a “connector portion”, such as the connector portion shown in Formula (I), which is part of the whole “connector”.

[0364] Formula I in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X1 and X2 are each selected independently from the following group: 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; and (ii) s, t, and v cannot all be 0 at the same time; Z represents the oligonucleotide moiety.

[0365] As will be further understood in the art, the exemplary compounds of this application comprise an entire connector located between the oligonucleotide moiety and the ligand moiety of these compounds. The entire connector then “connects” the oligonucleotide moiety and the ligand moiety to each other.

[0366] The entire linker is typically theoretically conceived as comprising one or more linker building blocks. For example, there exists a linker portion, described as the “connecting portion” of formula (I), located adjacent to the ligand portion and typically connecting the ligand portion directly or indirectly to the oligonucleotide portion via a branching point. The connecting portion shown in formula (I) is also commonly referred to as the “ligand arm” of the entire linker. Another linker portion may also exist between the oligonucleotide portion and the branching point (but not always), which is typically referred to as the “tethering portion” of the entire linker, “tethering” the oligonucleotide portion to the remainder of the coupling compound. Such “ligand arms” and / or “linker portions” and / or “tethering portions” can be conceived with reference to the linear and / or branched configurations described above.

[0367] As can be seen from the claims and the remainder of the patent specification, the scope of this application extends to linear or branched configurations, and there is no limitation on the number of individual ligands that may be present. Furthermore, based on prior art and the expertise of oligonucleotide chemists, those skilled in the art should also recognize that many structures can be used as linker sites.

[0368] The remaining portion of the entire connector (excluding the connector portion) listed in the claims and the remainder of the patent specification is represented by its chemical composition (Formula (I)), which the inventors consider to be unique to this application. However, more generally, these chemical compositions can be described as the “tethering portion” as described above, where the “tethering portion” is a part of the entire connector comprising the atomic group between Z (i.e., the oligonucleotide portion) and the connector portion, as shown in Formula (I).

[0369] The chain part of Formula I In the case of formula (I), the “chain part” comprises the group of atoms between Z (i.e., the oligonucleotide part) and the linker part.

[0370] In some embodiments, R1 is always hydrogen. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl.

[0371] In some embodiments, R2 is a hydroxyl group. In some embodiments, R2 is a halogen. In some embodiments, R2 is fluorine. In some embodiments, R2 is chlorine. In some embodiments, R2 is bromine. In some embodiments, R2 is iodine. In some embodiments, R2 is a nitro group.

[0372] In some embodiments, X1 is methylene. In some embodiments, X1 is oxygen. In some embodiments, X1 is sulfur.

[0373] In some embodiments, X2 is methylene. In some embodiments, X2 is oxygen. In some embodiments, X2 is sulfur.

[0374] In some implementations, m=3.

[0375] In some implementations, n=6.

[0376] In some embodiments, X1 is oxygen and X2 is methylene. In some embodiments, both X1 and X2 are methylene.

[0377] In some implementations, q=1, r=2, s=1, t=1, v=1. In other implementations, q=1, r=3, s=1, t=1, v=1.

[0378] In some implementations, R1 is always hydrogen, n=6, m=3, R2 is fluorine, X2 is methylene, v=1, t=1, s=1, X1 is methylene, q=1, r=2.

[0379] Therefore, in some embodiments, the exemplary compounds of this application comprise the following structures: Formula (IV) In some implementations, 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.

[0380] Therefore, in some embodiments, the exemplary compounds of this application comprise the following structures: Equation (II) Alternative chain components During the synthesis of the compounds in this application, alternative chain-linking structures may occur. In some embodiments, the alternative chain-linking portion has one or more atomic variations throughout the chain-linking portion of the connector compared to the chain-linking portion described anywhere herein.

[0381] In some embodiments, the alternative chain portion is a compound of formula (I) described anywhere herein, wherein R2 is a hydroxyl group.

[0382] In some implementations, R1 is always hydrogen, n=6, m=3, R2 is hydroxyl, X2 is methylene, v=1, t=1, s=1, X1 is methylene, q=1 and r=2.

[0383] Therefore, in some embodiments, the compounds of this application comprise the following structures: Formula (V) In some implementations, 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.

[0384] Therefore, in some embodiments, the compounds of this application comprise the following structures: Equation (III) Connector section With regard to formula (I), the “connector portion” shown in formula (I) comprises the group of atoms located between the chain portion and the ligand portion as described anywhere in this document.

[0385] In some implementation schemes: As shown in equation (I) anywhere in this document It is any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa): Formula (VIa) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and b is an integer from 2 to 5; or Formula (VIb) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and c and d are independent integers from 1 to 6; or Formula (VIc) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and e is an integer from 2 to 10.

[0386] In some implementations, the portion shown in formula (I) is: This is formula (VIa): Formula (VIa) in: A I The protecting group is hydrogen or a suitable hydroxyl group; a is 3; and b is an integer 3.

[0387] In some implementations, such as the portion shown in formula (I) anywhere herein: Equation (VII): Equation (VII) in: A I It is hydrogen; a is an integer of 2 or 3, preferably 3.

[0388] Other exemplary compounds of this application include a “connector portion” as shown in formula (I*), which is part of the whole “connector”.

[0389] Formula I* in: r and s are independent integers selected from 1 to 16; Z represents the oligonucleotide moiety.

[0390] As will be further understood in the art, the exemplary compounds of this application comprise an entire connector located between the oligonucleotide moiety and the ligand moiety of these compounds. The entire connector then “connects” the oligonucleotide moiety and the ligand moiety to each other.

[0391] A monolithic linker is typically conceived theoretically as comprising one or more linker building blocks. For example, there exists a linker portion, described as the “linker portion” of formula (I*), located adjacent to the ligand portion and typically connecting the ligand portion directly or indirectly to the oligonucleotide portion via a branch point. The linker portion shown in formula (I*) is also commonly referred to as the “ligand arm” of the entire linker. Another linker portion may also exist between the oligonucleotide portion and the branch point (but not always), typically referred to as the “tether portion” of the entire linker, “connecting” the oligonucleotide portion to the remainder of the coupling compound. Such “ligand arms” and / or “linker portions” and / or “tether portions” can be conceived with reference to the linear and / or branched configurations described above.

[0392] As can be seen from the claims and the remainder of the patent specification, the scope of this application extends to linear or branched configurations, and there is no limitation on the number of individual ligands that may be present. Furthermore, based on prior art and the expertise of oligonucleotide chemists, those skilled in the art should also recognize that many structures can be used as linker sites.

[0393] The remaining portion of the entire connector (excluding the connector portion) listed in the claims and the remainder of the patent specification is represented by its chemical composition (Formula (I)), which the inventors consider to be unique to this application. However, more generally, these chemical compositions can be described as the “tethering portion” as described above, where the “tethering portion” is a part of the entire connector comprising the atomic group between Z (i.e., the oligonucleotide portion) and the connector portion, as shown in Formula (I).

[0394] Chain section In the case of formula (I*), the “chain part” comprises the group of atoms between Z (i.e., the oligonucleotide part) and the linker part.

[0395] In some implementations, s is an integer selected from 4 to 12. In some implementations, s is 6.

[0396] In some implementations, r is an integer selected from 4 to 14. In some implementations, r is 6. In some implementations, r is 12.

[0397] In some implementations, r is 12 and s is 6.

[0398] Therefore, in some embodiments, the exemplary compounds of this application comprise the following structures: Equation (II*) In some implementations, r is 6 and s is 6.

[0399] Therefore, in some embodiments, the exemplary compounds of this application comprise the following structures: Equation (III*) Connector section With regard to formula (I*), the “connector portion” shown in formula (I) comprises the set of atoms located between the chain portion and the ligand portion as described anywhere in this document.

[0400] In some implementations, this part is shown as in formula (I*) anywhere herein: It is any one of formula (IV*), (V*) or (VI*), preferably formula (IV*): Formula (IV*) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and b is an integer from 2 to 5; or Formula (V*) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and c and d are independent integers from 1 to 6; or Formula (VI*) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and e is an integer from 2 to 10.

[0401] In some implementations, the portion shown in formula (I) is: For example (VIa*): Formula (VIa*) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is 3; and b is an integer 3.

[0402] In some implementations, as shown in formula (I) anywhere herein: This is formula (VII*): Equation (VII*) in: A I It is hydrogen; a is an integer of 2 or 3.

[0403] In some implementations, a=2. In some implementations, a=3. In some implementations, b=3.

[0404] In some implementations, the GalNAc ligand is contained in Figures 2 to 5 or Figure 6 In any of the linkers shown in (Formula XI), the “oligonucleotide” can be any nucleic acid disclosed herein. Therefore, the “oligonucleotide” can contain bonds other than phosphodiester bonds, such as one or more thiophosphate bonds. Preferably, the nucleic acid of this application is a double-stranded oligonucleotide as defined herein, and the linker is coupled to the second strand via a phosphodiester bond, more preferably to the 3' terminal region of the second strand.

[0405] In some implementations, the GalNAc ligand is contained in Figure 4 In the connector shown, the term "oligonucleotide" can be any nucleic acid disclosed herein. Therefore, an "oligonucleotide" can contain bonds other than phosphodiester bonds, such as one or more thiophosphate bonds. Preferably, the nucleic acid of this application is a double-stranded oligonucleotide as defined herein, and the connector is coupled to the second strand via a phosphodiester bond, more preferably to the 3' terminal region of the second strand.

[0406] In some implementations, the GalNAc ligand is contained in Figure 6In the linker shown in (Formula XI), the “oligonucleotide” can be any nucleic acid disclosed herein. Therefore, the “oligonucleotide” can contain bonds other than phosphodiester bonds, such as one or more thiophosphate bonds. Preferably, the nucleic acid of this application is a double-stranded oligonucleotide as defined herein, and the linker is coupled to the second strand tip and, more preferably, to the 3' terminal region of the second strand via phosphodiester bonds.

[0407] In some implementations, the GalNAc ligand is contained in Figures 2 to 5 or Figure 6 In any of the adapters shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified or unmodified second chain, the second chain comprising or consisting of any one of SEQ ID NO:553 to SEQ ID NO:828, preferably wherein the adapter is coupled to the 3' end region of the second chain (i.e., the 3' end region of any one of SEQ ID NO:553 to SEQ ID NO:828) by a phosphodiester bond.

[0408] In some implementations, the GalNAc ligand is contained in Figure 4 In the connector shown, “oligonucleotide” represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified or unmodified second strand, the second strand comprising or consisting of any one of SEQ ID NO:553 to SEQ ID NO:828, preferably wherein the connector is coupled to the 3' end region of the second strand (i.e., the 3' end region of any one of SEQ ID NO:553 to SEQ ID NO:828) via a phosphodiester bond.

[0409] In some implementations, the GalNAc ligand is contained in Figure 6 In the adapter shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified or unmodified second chain, the second chain comprising or consisting of any one of SEQ ID NO:553 to SEQ ID NO:828, preferably wherein the adapter is coupled to the 3' end region of the second chain (i.e., the 3' end region of any one of SEQ ID NO:553 to SEQ ID NO:828) by a phosphodiester bond.

[0410] In some implementations, the GalNAc ligand is contained in Figures 2 to 5 or Figure 6In any of the adapters shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified or unmodified second strand comprising or composed of any one of SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 or SEQ ID NO:772, preferably wherein the adapter is coupled to the 3' terminal region of the second strand (i.e., the 3' terminal region of any one of SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772) via a phosphodiester bond.

[0411] In some implementations, the GalNAc ligand is contained in Figure 4 In the adapter shown, “oligonucleotide” represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified or unmodified second strand, the second strand comprising or consisting of any one of SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772, preferably wherein the adapter is coupled to the 3' terminal region of the second strand (i.e. the 3' terminal region of any one of SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772) via a phosphodiester bond.

[0412] In some implementations, the GalNAc ligand is contained in Figure 5 In the adapter shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified or unmodified second strand, the second strand comprising or consisting of any one of SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772, preferably wherein the adapter is coupled to the 3' terminal region of the second strand (i.e., the 3' terminal region of any one of SEQ ID NO:580, SEQ ID NO:599, SEQ ID NO:715, SEQ ID NO:729 and SEQ ID NO:772) via a phosphodiester bond.

[0413] In some implementations, the GalNAc ligand is contained in Figures 2 to 5 or Figure 6In any of the adapters shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second chain comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably wherein the adapter is coupled to the 3' end region of the second chain (i.e., the 3' end region of any one of SEQ ID NO:1105 to SEQ ID NO:1380) via a phosphodiester bond.

[0414] In some implementations, the GalNAc ligand is contained in Figure 4 In the connector shown, “oligonucleotide” represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second chain comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably wherein the connector is coupled to the 3' end region of the second chain (i.e., the 3' end region of any one of SEQ ID NO:1105 to SEQ ID NO:1380) via a phosphodiester bond.

[0415] In some implementations, the GalNAc ligand is contained in Figure 6 In the adapter shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second chain comprising or consisting of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably wherein the adapter is coupled to the 3' end region of the second chain (i.e., the 3' end region of any one of SEQ ID NO:1105 to SEQ ID NO:1380) via a phosphodiester bond.

[0416] In some implementations, the GalNAc ligand is contained in Figures 2 to 5 or Figure 6 In any of the adapters shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second strand comprising or composed of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, preferably wherein the adapter is coupled to the 3' end region of the second strand (i.e., the 3' end region of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324) via a phosphodiester bond.

[0417] In some implementations, the GalNAc ligand is contained in Figure 4In the adapter shown, “oligonucleotide” represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second strand comprising or composed of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, preferably wherein the adapter is coupled to the 3' terminal region of the second strand (i.e., the 3' terminal region of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324) via a phosphodiester bond.

[0418] In some implementations, the GalNAc ligand is contained in Figure 6 In the linker shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second chain comprising or composed of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, preferably wherein the linker is coupled to the 3' end region of the second chain (i.e., the 3' end region of any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324) via a phosphodiester bond.

[0419] In some implementations, the GalNAc ligand is contained in Figures 2 to 5 or Figure 6 In the adapter shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second strand comprising any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, or composed of any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, wherein the second strand has the following structure in: T represents 2'Me ribose modification. B represents any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, and the first two basic nucleoside bases of the 5' terminal region. Z represents any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, representing the remaining 19 consecutive basic nucleosides.

[0420] In some implementations, the GalNAc ligand is contained in Figure 6 In the adapter shown in (Formula XI), "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second strand comprising any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, or comprising any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably comprising any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, wherein the second strand has the following structure in: T represents 2'Me ribose modification. B represents any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, and the first two basic nucleoside bases of the 5' terminal region. Z represents any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, representing the remaining 19 consecutive basic nucleosides.

[0421] In some implementations, the GalNAc ligand is contained in Figure 4 In the connector shown, "oligonucleotide" represents the nucleic acid of this application, wherein the nucleic acid of this application comprises a modified second strand comprising any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, or comprising any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, wherein the second strand has the following structure in: T represents 2'Me ribose modification. B represents any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, and the first two basic nucleoside bases of the 5' terminal region. Z represents any one of SEQ ID NO:1105 to SEQ ID NO:1380, preferably any one of SEQ ID NO:1132, SEQ ID NO:1151, SEQ ID NO:1267, SEQ ID NO:1281 and SEQ ID NO:1324, representing the remaining 19 consecutive basic nucleosides.

[0422] carriers and cells In one aspect, this application provides cells containing nucleic acids (e.g., repressive RNA [RNAi]) as described herein.

[0423] In one aspect, this application provides cells comprising the vector described herein.

[0424] In one aspect, this application provides a vector containing an oligonucleotide inhibitor (e.g., iRNA, such as siRNA).

[0425] Pharmaceutically acceptable compositions In one aspect, this application provides a pharmaceutical composition for inhibiting the expression of a target gene, the composition comprising an inhibitor disclosed herein, such as an oligomer, such as a nucleic acid.

[0426] The pharmaceutically acceptable composition may contain excipients and / or carriers.

[0427] Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth gum powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol. Alcohols; (11) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyesters, polycarbonates and / or polyanhydrides; (22) Fillers, such as peptides and amino acids; (23) Serum components, such as serum albumin, HDL and LDL; and (24) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0428] Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium carboxymethyl starch); and wetting agents (e.g., sodium dodecyl sulfate).

[0429] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and that do not react harmfully with nucleic acids may also be used to formulate the compositions of this application. Suitable pharmaceutically acceptable excipients include, but are not limited to: water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0430] Formulations for topical application of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents (e.g., alcohols), or solutions of nucleic acids in liquid or solid oily matrices. These solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and that do not cause adverse reactions with nucleic acids may be used.

[0431] In one embodiment, the nucleic acid or composition is administered in a non-buffered solution. In some embodiments, the non-buffered solution is saline or water. In other embodiments, the nucleic acid (e.g., an RNAi agent) is administered in a buffered solution. In these embodiments, the buffered solution may contain acetate, citrate, prolyl protein, carbonate, or phosphate, or any combination thereof. For example, the buffered solution may be phosphate-buffered saline (PBS).

[0432] dose The pharmaceutical composition of this application can be administered at a dose sufficient to inhibit gene expression or modify target expression or function. Generally, when the composition contains nucleic acid, the appropriate dose of the nucleic acid (e.g., siRNA) of this application is in the range of about 0.001 to about 200.0 mg / kg (recipient's body weight) / day, typically about 1 to 50 mg / kg / day. Typically, the appropriate dose of the nucleic acid (e.g., siRNA) of this application is in the range of about 0.1 mg / kg to about 5.0 mg / kg, for example about 0.3 mg / kg and about 3.0 mg / kg.

[0433] Repeated dosing regimens may include the periodic administration of therapeutic doses of nucleic acids (e.g., siRNA) (e.g., every other day or once a year). In some implementations, the frequency of administration of nucleic acids (e.g., siRNA) is approximately once a month to once a quarter (i.e., approximately once every three months).

[0434] In various embodiments, the nucleic acid (e.g., siRNA) reagent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg, or about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) reagent is administered at a dose of about 10 mg / kg to about 30 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) reagent is selected from about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) reagent is administered about once a week, once a month, once every two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg / kg to about 5.0 mg / kg. In some embodiments, the nucleic acid (e.g., siRNA) reagent is administered to the subject once a week. In some embodiments, the nucleic acid (e.g., siRNA) reagent is administered to the subject once a month. In some implementations, the subject is given a nucleic acid (e.g., siRNA agent) once every quarter (i.e., every three months).

[0435] After completing the initial treatment regimen, treatment can be administered at a lower frequency. For example, once a week or every two weeks for three months, followed by once a month for six months or a year, or even longer.

[0436] The pharmaceutical composition can be administered once daily, or in two, three, or more sub-dose doses at appropriate intervals throughout the day, or even delivered via continuous infusion or controlled-release formulations. In this case, the amount of nucleic acid (e.g., siRNA) contained in each sub-dose must be correspondingly smaller to achieve the total daily dose. Dosage units can also be formulated for delivery over several days, for example using conventional sustained-release formulations that continuously release nucleic acid (e.g., siRNA) over several days. Sustained-release formulations are well known in the art and are particularly suitable for delivering agents at specific sites, and can be used, for example, with the agents of this application. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0437] In other embodiments, a single dose of the pharmaceutical composition may be administered for an extended period, such that subsequent doses are administered at intervals not exceeding 3, 4, or 5 days, or at intervals not exceeding 1, 2, 3, or 4 weeks. In some embodiments of this application, a single dose of the pharmaceutical composition is administered once weekly. In other embodiments of this application, a single dose of the pharmaceutical composition is administered every two months. In some embodiments, siRNA is administered approximately once monthly to approximately once quarterly (i.e., approximately once every three months), or even once every 6 months or 12 months.

[0438] The effective dose and in vivo half-life of the individual nucleic acid (e.g., siRNA) covered by this application can be estimated using conventional methods known in the art or based on in vivo experiments using appropriate animal models.

[0439] The pharmaceutical composition of this application can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be local (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or blowing of powder or aerosol, including via a nebulizer); intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous administration (e.g., via an implanted device); or intracranial administration (e.g., via intraparenchymal, intrathecal, or intraventricular administration). In some preferred embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by subcutaneous injection.

[0440] In one implementation, nucleic acid (e.g., siRNA agent) is administered subcutaneously to the subject.

[0441] Inhibitors (such as nucleic acids, such as siRNA) can be delivered in a manner that targets specific tissues (e.g., particularly hepatocytes).

[0442] Methods for inhibiting gene expression or inhibiting target expression or function in vitro This application also provides a method for inhibiting the expression of the SLC25A5 gene in cells. The method involves contacting cells with a nucleic acid (e.g., an siRNA agent, such as a double-stranded siRNA agent) of this application, the amount of which effectively inhibits the expression of the SLC25A5 gene in the cells, thereby inhibiting the expression of the SLC25A5 gene in the cells. It should be noted that the nucleic acid "used to inhibit SLC25A5 expression" refers to a nucleic acid capable of inhibiting SLC25A5 expression, preferably as described below.

[0443] Contacting cells with nucleic acids (e.g., siRNA, such as double-stranded siRNA agents) can be performed in vitro or in vivo. For example, in vivo contacting cells with nucleic acids includes contacting cells or cell populations within a subject (e.g., a human subject) with nucleic acids (e.g., siRNA). In vitro and in vivo methods of cell contact can also be combined. As mentioned above, cell contact can be direct or indirect. Furthermore, cell contact can be achieved by targeting ligand portions, including any ligand portions described herein or known in the art. In a preferred embodiment, the targeting ligand portion is a carbohydrate portion (e.g., GalNAc3 ligand) that guides the siRNA agent to a target site, or any other ligand portion.

[0444] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition.

[0445] In some embodiments of the method of this application, the expression or activity of the gene or inhibitory target is inhibited by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or inhibited below the detection level of the assay, preferably by qPCR assay as described herein and / or by transfection of siRNA into target cells. In some embodiments, the method includes clinically relevant inhibition of target gene expression, for example, demonstrated by clinically relevant outcomes following treatment of a subject with an agent to reduce gene expression and / or target activity.

[0446] In some embodiments, when the nucleic acid of this application is transfected into cells, it inhibits the expression of the SLC25A5 gene with an IC50 value below a specified threshold. In some embodiments, the threshold may be 2500 pM, 2000 pM, 1900 pM, 1800 pM, 1700 pM, 1600 pM, 1500 pM, 1400 pM, 1300 pM, 1200 pM, 1100 pM, 1000 pM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM, preferably determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0447] In a preferred embodiment, when the nucleic acid of this application is transfected into cells, its IC50 value for inhibiting SLC25A5 gene expression is less than 2500 pM. In a more preferred embodiment, when the nucleic acid of this application is transfected into cells, its IC50 value for inhibiting SLC25A5 gene expression is less than 1000 pM. In a further preferred embodiment, when the nucleic acid of this application is transfected into cells, its IC50 value for inhibiting SLC25A5 gene expression is less than 500 pM. In a most preferred embodiment, when the nucleic acid of this application is transfected into cells, its IC50 value for inhibiting SLC25A5 gene expression is less than 100 pM.

[0448] SLC25A5 gene inhibition can be quantified using the following methods: Huh7 cells (a human hepatocyte-derived cell line obtained from the JCRB cell bank) were cultured at 37°C and 5% CO2 in DMEM medium supplemented with 10% FBS and 1% non-essential amino acids. Subsequently, cells were transfected with siRNA duplexes targeting SLC25A5 mRNA or negative control siRNA (siRNA-control; sense strand 5'-GCCTGTACCAAGGCTTTAA-3' (SEQ ID NO: 1383), antisense strand 5'-TTAAAGCCTTGGTACAGGC-3' (SEQ ID NO: 1382)) using six 10-fold serial dilutions, resulting in duplex concentrations ranging from 3 nM to 0.03 pM. Transfection was performed by adding 9.7 µL of Opti-MEM (ThermoFisher) and 0.3 µL of Lipofectamine RNAiMAX (ThermoFisher) to each 10 µL siRNA duplex. The mixture was incubated at room temperature for 15 minutes and then added to 100 µL of complete growth medium containing 20,000 Huh7 cells. Cells were incubated at 37°C / 5% CO2 for 24 hours, followed by total RNA purification using the RNeasy 96 kit (Qiagen). Each duplex was tested in a single experiment via transfection in repeat wells.

[0449] cDNA synthesis can be performed using the FastKing RT (containing gDNase) kit (Tiangen). Real-time quantitative PCR (qPCR) can be performed on an ABI Primism 7900HT or ABI QuantStudio 7 using specific primers for human SLC25A5 (forward: ACTGACATCATGTACACAGGCAC (SEQ ID NO:1384), reverse: ACCCATGCCTCTGAGAACATT (SEQ ID NO:1385)) and human GAPDH (forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:1386), reverse: GAGATGGTGATGGGATTTC (SEQ ID NO:1387)) using the SensiFAST SYBR Hi-ROX kit (Meridian).

[0450] qPCR can be repeated for cDNA from each well, and the average cycle threshold (Ct) can be calculated. Comparison of Ct ( The Ct method calculates relative SLC25A5 expression levels based on the average Ct value, normalized to GAPDH, and relative to untreated cells. The maximum percentage inhibition and IC50 value of SLC25A5 expression can be calculated using the four-parameter (variable slope) model in GraphPad Prism 9.

[0451] In some embodiments, when the nucleic acid of this application is transfected into cells, its pEC50 value for inhibiting SLC25A5 gene expression is lower than 5, 6, 7, 8, 9 or 10, preferably determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0452] For this purpose, Huh7 cells (a human hepatocyte-derived cell line obtained from the JCRB cell bank) were cultured at 37°C, 5% CO2, and 95% humidity in DMEM medium supplemented with 10% FBS and 1% non-essential amino acids. Cells were transfected using double-stranded siRNAs targeting SLC25A5 mRNA or negative control siRNAs (siRNA-control; sense strand 5'-GCCTGTACCAAGGCTTTAA-3' (SEQ ID NO:1383), antisense strand 5'-TTAAAGCCTTGGTACAGGC-3' (SEQ ID NO:1382)) using a 6-point logarithmic dose-response curve, with final concentrations ranging from 3 nM to 0.03 pM. Transfection was performed by diluting Lipofectamine RNAiMAX (ThermoFisher) at a ratio of 48.5:1.5 in Opti-MEM (ThermoFisher) medium. Add an equal volume of siRNA to this solution and dilute to the desired concentration with phosphate-buffered saline. Incubate the lipofectamine RNAiMAX and siRNA mixture at room temperature for 15 minutes, then add 20 µL of the mixture to the wells of a 96-well plate. Isolate Huh7 cells from the culture flask using trypsin and resuspend them at a density of 200,000 cells / mL. Add 100 µL of the Huh7 cell suspension to each well of the 96-well plate containing the siRNA. Incubate the cells at 37°C, 5% CO2, and 95% humidity for 24 hours. Each siRNA can be tested in three wells over two days, for a total of six replicates.

[0453] Intracellular RNA can be isolated using the Rneasy kit (Qiagen) according to the manufacturer's instructions. cDNA synthesis can be performed using the FastKing RT (with gDNase) kit (Tiangen). Target cDNA can be quantified by qPCR using specific primers for human SLC25A5 (forward: ACTGACATCATGTACACAGGCAC (SEQ ID NO:1384), reverse: ACCCATGCCTCTGAGAACATT (SEQ ID NO:1385)) and human GAPDH (forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:1386), reverse: GAAGATGGTGATGGGATTTC (SEQ ID NO:1387)) using the SensiFAST SYBR Hi-ROX kit (Meridian).

[0454] qPCR can be repeated for cDNA from each well, and the average Ct value can be calculated. The Ct value can be compared using... The Ct method was used to calculate the relative expression level of SLC25A5 based on the average Ct value, normalized to GAPDH, and relative to untreated cells. The maximum percentage inhibition of SLC25A5 expression and the pEC50 value (EC50 value minus log) could be calculated using a four-parameter (variable slope) model in NumPy (Python). 10 ).

[0455] Alternatively or additionally, suppression of SLC25A5 gene expression can be characterized by a decrease in the average relative expression of the SLC25A5 gene.

[0456] In some embodiments, when cells are transfected with 0.1 nM of the nucleic acid of this application, the mean relative expression of SLC25A5 is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5 or 0.4, preferably as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0457] In some embodiments, when cells are transfected with 5 nM of the nucleic acid of this application, the mean relative expression of SLC25A5 is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3, preferably as determined by qPCR, more preferably by reverse transcriptase (RT)-qPCR, as described herein.

[0458] The average relative expression level of the SLC25A5 gene can be quantified using the following methods: Huh7 cells (human hepatocyte-derived cell line, obtained from the JCRB cell bank) were cultured at 37°C and 5% CO2 in DMEM medium supplemented with 10% FBS. Double-stranded siRNA targeting SLC25A5 mRNA or negative control siRNA (siRNA-control: sense strand 5'-GCCTGTACCAAGGCTTTAA-3' (SEQ ID NO: 1383), antisense strand 5'-TTAAAGCCTTGGTACAGGC-3' (SEQ ID NO: 1382)) were transfected into the cells at final concentrations of 5 nM and 0.1 nM, respectively. Transfection was performed by adding 9.7 µL of Opti-MEM (ThermoFisher) and 0.3 µL of Lipofectamine RNAiMAX (ThermoFisher) to each 10 µL siRNA double-stranded sample. The mixture was incubated at room temperature for 15 minutes and then added to 100 µL of complete growth medium containing 20,000 Huh7 cells. Cells were incubated at 37°C / 5% CO2 for 24 hours, and then total RNA was purified using the Rneasy 96 kit (Qiagen). Each duplex was detected in two independent experiments via transfection in repeat wells.

[0459] cDNA synthesis can be performed using the FastKing RT (containing gDNase) kit (Tiangen). It can be performed on an ABI Prism 7900HT or ABI QuantStudio 7 using primers targeting human SLC25A5 (forward: ACTGACATCATGTACACAGGCAC (SEQ ID NO:1384), reverse: ACCCATGCCTCTGAGAACATT (SEQ ID NO:1385)) and human GAPDH (forward: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:1386), reverse: GAGATGGTGATGGGATTTC (SEQ ID NO:1387)) using the SensiFAST SYBR Hi-ROX kit (Meridian).

[0460] qPCR can be repeated for cDNA from each well, and the average Ct can be calculated. Comparisons of Ct (...) can be made. The Ct method calculates the relative SLC25A5 expression level based on the average Ct value, normalizes GAPDH, and is relative to untreated cells.

[0461] Compared to a suitable control, the inhibition of SLC25A5 gene expression can be demonstrated by a reduction in the amount of mRNA in the target SLC25A5 gene.

[0462] In other implementations, the suppression of SLC25A5 gene expression can be assessed based on a reduction in parameters related to gene expression function, such as protein expression or signaling pathways.

[0463] Methods for treating or preventing diseases related to gene expression / target functional expression This application also provides a method for reducing or inhibiting gene expression in cells, or reducing the expression or function of a target gene, using the nucleic acid (e.g., siRNA) of this application or a composition containing the nucleic acid (e.g., siRNA) of this application. The method includes contacting cells with the nucleic acid (e.g., dsiRNA) of this application and maintaining the cells for a sufficient time to allow the degradation of the mRNA transcript of the gene, thereby inhibiting the expression of the gene in the cells. The reduction in the expression or function of the target gene can be assessed by any method known in the art. In a preferred embodiment, the gene is... SLC25A5 .

[0464] In the method of this application, the cells can be contacted in vitro or in vivo, that is, the cells can be in the body of the subject.

[0465] Cells suitable for treatment using the method of this application can be any cell expressing a target gene associated with a metabolic disease or condition, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or diseases or conditions associated with adipogenesis, and / or adipogenesis.

[0466] The in vivo method of this application may include administering to a subject a composition containing a nucleic acid (e.g., siRNA) of this application, wherein the nucleic acid (e.g., siRNA) comprises a nucleoside sequence complementary to at least a portion of the RNA transcript of the mammalian SLC25A5 gene to be treated.

[0467] This application also provides methods for treating subjects in need. The treatment methods of this application include administering, in a therapeutically effective amount, the nucleic acid of this application (e.g., siRNA), such as a nucleic acid targeting a gene (e.g., siRNA), or a pharmaceutical composition containing a nucleic acid targeting a gene to a subject (e.g., a subject who benefits from reduced or suppressed gene expression and / or target expression and / or function).

[0468] Nucleic acids (such as the siRNA of this application) can be administered in the form of "free" nucleic acids or "free" siRNA, in the presence of a drug-free composition. The naked nucleic acid can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, prolyl, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmotic pressure of the buffer solution can be adjusted to suit its administration to the subject.

[0469] Alternatively, the nucleic acid (e.g., siRNA) of this application may be administered in the form of a pharmaceutical composition, such as a dsiRNA liposome formulation.

[0470] In one embodiment, the method includes administering the composition described herein to reduce the expression of a target gene, for example, for a reduction lasting about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the reduction in target gene expression lasts for a longer period, for example, at least about two, three, four days, or longer, for example, about one week, two weeks, three weeks, four weeks, or longer, for example, about one month, two months, or three months.

[0471] Therapeutic doses of nucleic acid (e.g., siRNA) may be administered to the subject, for example, from about 0.01 mg / kg to about 200 mg / kg.

[0472] Nucleic acids (e.g., siRNA) can be administered periodically via intravenous infusion for a period of time. In some embodiments, treatment can be administered at a lower frequency after an initial treatment regimen. Administration of siRNA can reduce the level of the gene product of the target gene (e.g., in the patient's cells or tissues) by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the detection level of the assay method used. In some embodiments, administration results in clinical stability, or preferably clinically relevant remission, of at least one sign or symptom of the gene-related disease.

[0473] Alternatively, nucleic acids (e.g., siRNA) can be administered subcutaneously, i.e., by subcutaneous injection. The required daily dose of nucleic acid (e.g., siRNA) can be delivered to the subject using one or more injections. Injections can be repeated over a period of time. Administration can be repeated periodically. In some embodiments, treatment can be administered at a lower frequency after the initial treatment regimen. Repeated dosing regimens may include the administration of a therapeutic dose of nucleic acid periodically (e.g., every other day or once a year). In some embodiments, the frequency of nucleic acid administration is approximately once a month to once a quarter (i.e., once every three months).

[0474] combination therapy The inhibitors of this application may be used in combination with other therapeutic agents for therapeutic purposes, particularly for the treatment of any metabolic disease or condition disclosed herein.

[0475] In some embodiments, the inhibitors of this application, such as any siRNA molecules disclosed herein, may be combined with GLP-1 agonists (including, but not limited to, GLP-1 / GIP dual agonists, GLP-1 / FGF21 dual agonists, GLP-1 / GCGR dual agonists, and GLP-1 / GIP / GCGR triple agonists) and / or THR-β agonists.

[0476] In other words, in some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with GLP-1 agonists to treat metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with GLP-1 agonists to treat fatty liver, particularly NAFLD and / or NASH.

[0477] As used herein, the term "GLP-1 agonist" refers to a compound that fully or partially activates the human GLP-1 receptor. Therefore, this term is equivalent to the term "GLP-1 receptor agonist" as used in other documents. The term "GLP-1 agonist," as well as the specific GLP-1 agonists described herein, also encompass their salt forms.

[0478] Therefore, a GLP-1 agonist should possess "GLP-1 activity," which refers to the ability of a compound (i.e., a GLP-1 analog or a compound containing a GLP-1 analog) to bind to the GLP-1 receptor and initiate a signal transduction pathway, thereby producing insulinotropic effects or other physiological effects known in the art. In some embodiments, the "GLP-1 agonist" binds to the GLP-1 receptor, for example, by an affinity constant (K... D The agonist or its activating receptor titer (EC50) is less than 1 mM, for example less than 100 nM, as measured by methods known in the art (see, for example, WO 98 / 08871), and exhibits insulin-promoting activity, wherein the insulin-promoting activity can be measured by in vivo or in vitro assays known to those skilled in the art. For example, a GLP-1 agonist can be administered to animals with elevated blood glucose (e.g., using an intravenous glucose tolerance test (IVGTT). Those skilled in the art will be able to determine the appropriate glucose dose and appropriate blood collection protocol for the IVGTT, for example, depending on the animal species) and the change in plasma insulin concentration over time can be measured. Suitable assays have been described, for example, in WO2015 / 155151.

[0479] The term half-maximal effective concentration (EC50) generally refers to the concentration at which a response is induced at an intermediate level between the baseline and the maximum value, according to a dose-response curve. EC50 is used as a measure of compound potency, representing the concentration at which 50% of its maximum effect is observed. Because GLP-1 agonists containing the substituents described herein exhibit albumin-binding effects, it is essential to determine the presence of human serum albumin.

[0480] The in vitro potency of the GLP-1 agonist can be determined and the EC50 determined in the absence of human serum albumin (HSA) as described in Example 29 of WO2015 / 155151. A lower EC50 value indicates better potency. In one embodiment, the determined potency (EC50) (without HSA) is 5-1000 pM, for example 10-750 pM, 10-500 pM, or 10-200 pM. In one embodiment, the EC50 (without HSA) is at most 500 pM, for example at most 300 pM, for example at most 200 pM.

[0481] In one implementation, EC50 (excluding HSA) is equivalent to human GLP-1 (7-37).

[0482] In one embodiment, EC50 (excluding HSA) is at most 50 pM. In another such embodiment, EC50 is at most 40 pM, for example, at most 30 pM, for example, at most 20 pM, for example, at most 10 pM. In one embodiment, EC50 is about 10 pM.

[0483] Alternatively, the in vitro potency assay (including HSA) of Example 29 of WO2015 / 155151 can be used to measure the binding of GLP-1 agonists to albumin. An increase in the in vitro EC50 value in the presence of serum albumin reflects an affinity for serum albumin.

[0484] In one embodiment, the measured potency (EC50) (containing 1% HSA) is 5-1000 pM, for example 100-750 pM, 200-500 pM, or 100-400 pM. In one embodiment, the EC50 (containing 1% HSA) is at most 750 pM, for example at most 500 pM, for example at most 400 pM, for example at most 300 pM, or for example at most 250 pM.

[0485] If necessary, the fold change relative to a known GLP-1 receptor agonist can be calculated as EC50 (test analog) / EC50 (known analog), and if the ratio is, for example, 0.5–1.5 or 0.8–1.2, the potency is considered equivalent.

[0486] In one embodiment, the EC50 (HSA-free) is equivalent to the potency of liraglutide. In another embodiment, the EC50 (HSA-free) is equivalent to the potency of smegglutide.

[0487] In some embodiments, the GLP-1 agonist is a GLP-1 analog, optionally containing “one substituent”. As used herein, the term “analyte” to refer to a GLP-1 peptide (hereinafter “peptide”) means a peptide in which at least one amino acid residue has been substituted by another amino acid residue, and / or at least one amino acid residue has been deleted, and / or at least one amino acid residue has been added, and / or at least one amino acid residue has been modified. Such addition or deletion of amino acid residues can occur at the N-terminus and / or C-terminus of the peptide.

[0488] In some embodiments, the term "GLP-1 analog" or "GLP-1 analogue" as used herein refers to a peptide or compound of a variant of human glucagon-like peptide-1 (GLP-1 (7-37)). The sequence of GLP-1 (7-37) is HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 1388). In some embodiments, the term "variant" refers to a compound comprising one or more amino acid substitutions, deletions, additions, and / or insertions.

[0489] In one embodiment, the GLP-1 agonist has at least 60%, 65%, 70%, 80%, or 90% sequence identity with GLP-1 (7-37) over the entire length of GLP-1 (7-37).

[0490] Generally, the term "GLP-1 agonist" is intended to cover GLP-1 agonists and any pharmaceutically acceptable salts, amides, or esters thereof. In some embodiments, the composition comprises a GLP-1 agonist or a pharmaceutically acceptable salt, amide, or ester thereof. In some embodiments, the composition comprises a GLP-1 agonist and one or more pharmaceutically acceptable counterions.

[0491] In some embodiments, the inhibitor of this application, particularly the siRNA molecule of this application, may be administered in combination with a GLP-1 agonist selected from one or more GLP-1 agonists disclosed below: WO93 / 19175, WO96 / 29342, WO98 / 08871, WO99 / 43707, WO99 / 43706, WO99 / 43341, WO99 / 43708, WO2005 / 027 978, WO2005 / 058954, WO2005 / 058958, WO2006 / 005667, WO2006 / 037810, WO2006 / 037811, WO2006 / 097537, WO2006 / 097538, WO2008 / 023050, WO2009 / 030738, WO2009 / 030771, WO2009 / 030774 and WO2021 / 219710, the entire contents of which are incorporated herein by reference.

[0492] In some embodiments, the inhibitor of this application, particularly the siRNA molecule of this application, may be administered in combination with a GLP-1 agonist selected from the group consisting of: Dulaglutide (Trulicity®), exenatide (Byetta®), exenatide extended-release (Bydureon®), liraglutide (Victoza®), lixisenatide (Adlyxin®), semaglutide injection (Ozempic®), and semaglutide tablets (Rybelsus®).

[0493] In some embodiments, the GLP-1 agonist is semaglutide having the following molecular formula: N-ε26-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetamido]ethoxy}ethoxy)acetyl] [Aib8,Arg34]GLP-1 (7-37).

[0494] As used herein, the term "GLP-1 agonist" also encompasses dual or triple agonists capable of activating more than one receptor. These dual or triple agonists can be molecules capable of activating the GLP-1 receptor and at least one other receptor. In some embodiments, the dual or triple agonist can be a chimeric molecule comprising a first portion that activates the GLP-1 receptor and other portions that activate other receptors. In some embodiments, the GLP-1 agonist is a dual or triple agonist that, in addition to the GLP-1 receptor, activates one or more of the following: glucose-dependent insulinotropic peptide (GIP) receptor, fibroblast growth factor 21 (FGF21) receptor, and glucagon receptor (GCGR).

[0495] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be combined with a GLP-1 / GIP dual agonist for the treatment of metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be combined with a GLP-1 / GIP dual agonist for the treatment of fatty liver, particularly NAFLD and / or NASH.

[0496] As used in the context of this application, the term "GLP-1 / GIP dual agonist" refers to a substance or ligand capable of activating both the GLP-1 receptor and the glucose-dependent insulinotropic peptide (GIP) receptor. GLP-1 / GIP receptor co-agonists and their potential medical uses have been described in numerous patent applications, such as WO 2010 / 011439, WO 2013 / 164483, WO 2014 / 192284, WO 2015 / 067715, WO 2015 / 022420, WO 2015 / 086728, WO 2015 / 086729, WO 2016 / 111971, WO 2020 / 023386, US 9745360, US 2014 / 162945, US 2014 / 0357552, WO 2021 / 150673, WO 2021 / 260530, WO 2022 / 018185, and WO 2022 / 079639, which is incorporated into this article in its entirety by reference.

[0497] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with the GLP-1 / GIP dual agonist telpokines (Mounjaro®) for the treatment of metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with the GLP-1 / GIP dual agonist telpokines (Mounjaro®) for the treatment of fatty liver, particularly NAFLD and / or NASH.

[0498] As used herein, “telpokinesin” refers to the GLP-1 / GIP dual agonist peptide described in U.S. Patent No. 9,474,780, CAS Registry No. 2023788-19-2. Telpokinesin is described in Example 1 of U.S. Patent No. 9,474,780, and its sequence is as follows: YX1EGTFTSDYSIX2LDKIAQKAFVQWLMGGPSSGAPPPS(SEQ ID NO: 1389), Where X1 is α-aminoisobutyric acid (Aib); X2 is Aib; K at position 20 is connected to (2-[2-(2-aminoethoxy)-ethoxy]-acetyl)2-(γGlu)1-CO-(CH2) via the ε-amino group of the K side chain. 18 -CO2H coupling is used for chemical modification; and the C-terminal amino acid is amidated to a C-terminal primary amide.

[0499] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with a GLP-1 / FGF21 dual agonist for the treatment of metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with a GLP-1 / FGF21 dual agonist for the treatment of fatty liver, particularly NAFLD and / or NASH.

[0500] As used in the context of this application, the term "GLP-1 / FGF21 dual agonist" refers to a substance or ligand capable of activating both the GLP-1 receptor and the fibroblast growth factor 21 (FGF21) receptor. Preferably, the GLP-1 / FGF21 dual agonist is a chimeric molecule comprising a GLP-1 agonist and an FGF21 molecule as defined above, or a functionally active variant or analog thereof. In some embodiments, the GLP-1 / FGF21 dual agonist may also comprise an antibody Fc region.

[0501] GLP-1 / FGF21 receptor co-agonists and their potential medical uses have been described in several patent applications, such as WO 2010 / 142665, WO2014 / 037373, WO 2018 / 115401, WO 2018 / 166461, WO 2019 / 243557 and WO2022 / 002408, all of which are incorporated herein by reference.

[0502] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be combined with a GLP-1 / GCGR dual agonist for the treatment of metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be combined with a GLP-1 / GCGR dual agonist for the treatment of fatty liver disease, particularly NAFLD and / or NASH.

[0503] As used in the context of this application, the term "GLP-1 / GCGR dual agonist" refers to a substance or ligand capable of activating both the GLP-1 receptor and the glucagon receptor (GCGR). GLP-1 / GCGR receptor co-agonists and their potential medical uses have been described in numerous patent applications, such as WO 2008 / 101017, WO 2009 / 155258, WO 2011 / 075393, WO2011 / 160630, WO 2014 / 056872, WO 2014 / 091316, WO 2015 / 086733, WO 2017 / 181452, WO2018 / 100174, WO 2019 / 030268, WO 2019 / 060660, and WO 2023 / 006923, all of which are incorporated herein by reference.

[0504] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with the GLP-1 / GCGR dual agonist survodutide (BI 456906) for the treatment of metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with the GLP-1 / GCGR dual agonist survodutide (BI 456906) for the treatment of fatty liver disease, particularly NAFLD and / or NASH.

[0505] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with a GLP-1 / GIP / GCGR triple agonist for the treatment of metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with a GLP-1 / GIP / GCGR dual agonist for the treatment of fatty liver disease, particularly NAFLD and / or NASH.

[0506] As used in the context of this application, the term "GLP-1 / GIP / GCGR triple agonist" refers to a substance or ligand capable of activating the GLP-1 receptor, GIP receptor, and glucagon receptor (GCGR). GLP-1 / GIP / GCGR receptor co-agonists and their potential medical uses have been described in numerous patent applications, such as WO 2014 / 096150, WO 2015 / 067716, WO 2019 / 125292, WO 2022 / 090447, and WO 2022 / 268029, the entire contents of which are incorporated herein by reference.

[0507] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with Retatrutid (LY-3437943) to treat metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with Retatrutid (LY-3437943) to treat fatty liver diseases, particularly NAFLD and / or NASH.

[0508] Those skilled in the art know methods for formulating GLP-1 agonists (including dual and triple agonists) for use in any suitable route of administration. In some embodiments, the GLP-1 agonist may be administered orally or by injection, such as subcutaneously.

[0509] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with THR-β agonists to treat metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be used in combination with THR-β agonists to treat fatty liver, particularly NAFLD and / or NASH.

[0510] As used herein, the term "THR-β agonist" refers to a compound that fully or partially activates the human thyroid hormone receptor β. The term THR-β agonist, as well as the specific THR-β agonists described herein, also encompasses their salt forms.

[0511] Various THR-β agonists have been described in this field and summarized by Zucchi (Thyroid Hormone Analogues: An Update, Thyroid. August 2020; 30(8): 1099–1105) et al., the full text of which is incorporated herein by reference.

[0512] In some embodiments, the THR-β agonist administered in combination with the inhibitor of this application is MGL-3196 (remetiro), KB-2115 (iloterol), GC-1 (sobetirome), or MB07344 / VK2809, as described by Zucchi.

[0513] In some embodiments, the THR-β agonist is MGL-3196 (remetitrol) or any compound disclosed in WO 2014 / 043706, the entire contents of which are incorporated herein by reference. Remetitrol is a selective thyroid hormone receptor (THR) β agonist. Publications relating to remetitrol can be found in U.S. Patent No. 9,266,861, U.S. Patent Application Serial No. 16 / 343,065, and PCT Application Serial No. PCT / US2019 / 040276, the entire contents of which are incorporated herein by reference.

[0514] In some embodiments, the THR-β agonist is any compound disclosed in KB-2115 (eprotirome) or WO2007 / 110226 or WO 2009 / 077147, the entire contents of which are incorporated herein by reference.

[0515] Those skilled in the art know methods for formulating THR-β agonists for any suitable route of administration.

[0516] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be combined with GLP-1 agonists (such as any of the GLP-1 agonists disclosed herein, including dual or triple agonists) and THR-β agonists (such as any of the THR-β agonists disclosed herein) for the treatment of metabolic diseases or conditions. In one specific embodiment, the inhibitors of this application, particularly the siRNA molecules disclosed herein, can be combined with GLP-1 agonists (such as any of the GLP-1 agonists disclosed herein, including dual or triple agonists) and THR-β agonists (such as any of the THR-β agonists disclosed herein) for the treatment of fatty liver, particularly NAFLD and / or NASH.

[0517] The inhibitors of this application, particularly the siRNA molecules disclosed herein, may alternatively or additionally be administered in combination with and / or co-administered with one or more of the following: an amylin receptor agonist (e.g., pramlintide), and / or a dual amylin + calcitonin receptor agonist, and / or a glucagon receptor agonist, and / or an FXR receptor agonist (e.g., cilofexor or obeticholic acid). FGF-21 analogs or FGF-21 receptor agonists (e.g., eruxifermin), FGF-19 analogs or FGF-19 receptor agonists (e.g., aldafermin), and / or galactoglobulin 3 inhibitors (e.g., belapectin), and / or PPARα agonists (e.g., erafibrano), and / or PPARγ agonists (e.g., pioglitazone or rosiglitazone), and / or mixed PPARα and / or δ and / or γ agonists, and / or pan-PPARαδγ agonists (e.g., lanilano), and / or acetyl-CoA dehydrogenase. Saturase activators, and / or ASK1 inhibitors (e.g., seloselte) and / or LOXL2 inhibitors (e.g., simtuzumab) and / or dual CCR2 / 5 inhibitors (e.g., cineviro) and / or inhibitors of enzymes in the de novo lipogenesis (DNL) pathway, including citrate / isocitrate carrier (CIC), ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), and / or inhibitors of enzymes in the cholesterol biosynthesis pathway (e.g., HMGCoA reductase inhibitors, such as atorvastatin); preferably for the treatment of any metabolic disease or condition disclosed herein, more preferably for the treatment of NAFLD.

[0518] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, may be used in combination with any of the molecules disclosed in Figure 1 of Nathani and Bansal (Gastroenterol Hepatol (NY). 2023 Jul; 19(7): 371–381; incorporated herein by reference) for the treatment of any metabolic disease or condition disclosed herein, more preferably for the treatment of NAFLD, wherein the molecules are particularly one or more of the following: THR-β agonists (e.g., remesmetirom, VK2809, or TERN-501), PPAR agonists (e.g., Lanifibranor, Saroglitazar, or Elafibranor), GLP-1 agonists (e.g., liraglutide, semaglutide, or tirzepatide), and CCR2 / 5 inhibitors (e.g., cinnickivir). Antigens such as roxostat, ASK1 inhibitors (e.g., selonsertib), ACC inhibitors (e.g., filsocostat or PF-05221304), SCD inhibitors (e.g., aramchol), FGF21 analogs (e.g., iluxivmin or pegbelfermin), galactoglobulin-3 inhibitors (e.g., belapectin), LOXL2 inhibitors (e.g., simtuzumab), FXR agonists (e.g., obeticholic acid, tropifexor, cilofaxor, EDP-305, or MET409), and / or FGF19 analogs (e.g., aldafermina).

[0519] In some embodiments, the inhibitors of this application, particularly the siRNA molecules disclosed herein, may be combined with any molecules disclosed by Batchuluun et al. (Nat Rev Drug Discov, 2022, 21(4):283-305. doi: 10.1038 / s41573-021-00367-2.; incorporated herein by reference) for the treatment of any metabolic disease or condition disclosed herein, more preferably for the treatment of NAFLD, wherein the molecules are particularly one or more of the following: citrate / isocitrate carrier (CIC) inhibitors (e.g., benzotrimethylammonium ester, CPTI-1 or CPTI-2), ATP-citrate lyase (ACLY) inhibitors (e.g., bempedoic acid, hydroxycitrate, BMS-303141, emodin derivatives, furanylcarboxylic acid ester derivatives, MEDICA, etc.). 16. SB-204990 or NDI-091143), acetyl-CoA carboxylase (ACC) inhibitors (e.g., filsocostat, PF-05221304, PF-05175157, MK-4074, A-908292, carboxamide derivative-1k, CP-640186, monocyclic derivative-1q, ND-654, ND-646, olefin derivative-2e, (S)-9c, soraphen A). A) TOFA or WZ66) and / or fatty acid synthase (FAS) inhibitors (e.g., orlistat, TVB-2640, FT-4101, BI-99179, cerulenin, C75, Fasnall, GSK2194069, IPI-9119, MP-ML-24-N1, or TVB-3166).

[0520] The combination therapy or "combination" described herein includes co-administration of the inhibitor of this application (particularly the siRNA of this application) with one or more other therapeutic agents (preferably one or more other therapeutic agents disclosed above). The inhibitor of this application may be administered before, after, or simultaneously with one or more other therapeutic agents.

[0521] In some embodiments, the inhibitor of this application, particularly the siRNA of this application, can be co-administered with a GLP-1 agonist (including a GLP-1 / GIP dual agonist, a GLP-1 / FGF21 dual agonist, a GLP-1 / GCGR dual agonist, and a GLP-1 / GIP / GCGR triple agonist) and / or a THR-β agonist, wherein the inhibitor of this application can be administered before, after, or simultaneously with the GLP-1 agonist and / or the THR-β agonist.

[0522] In one exemplary embodiment, co-administration includes administering the siRNA of this application and any GLP-1 agonist disclosed herein or incorporated herein by reference (including GLP-1 / GIP dual agonists, GLP-1 / FGF21 dual agonists, GLP-1 / GCGR dual agonists, and GLP-1 / GIP / GCGR triple agonists). In another exemplary embodiment, co-administration includes administering the siRNA of this application and the GLP-1 agonist smegglutide. In yet another exemplary embodiment, co-administration includes administering the siRNA of this application and the GLP-1 / GIP dual agonist terzapetide.

[0523] In another exemplary embodiment, co-administration includes administration of the siRNA of this application and any THR-β agonist disclosed herein or incorporated herein by reference. In another exemplary embodiment, co-administration includes administration of the siRNA of this application and the THR-β agonist retinotirol.

[0524] In another exemplary embodiment, the combined administration includes administration of the siRNA of this application, the GLP-1 agonist smegglutide or telpolide, and the THR-β agonist remetidine.

[0525] The combination therapy of this disclosure, comprising the inhibitor of this application and at least one other therapeutic agent, can be administered via any route. In some embodiments, the inhibitor of this application and at least one other therapeutic agent can be delivered orally, subcutaneously, intravenously, intranasally, percutaneously, intraperitoneally, intramuscularly, intrapulmonaryly, vaginally, rectally, or intraocularly. In exemplary embodiments, the inhibitor of this application can be administered intravenously (IV) and / or subcutaneously, and the GLP-1 agonist can be administered subcutaneously.

[0526] In one aspect, this application can be applied to the compounds, methods, compositions, or uses described in the statements numbered 1-101 below, wherein any chemical formula mentioned in statements 1-101 refers only to those chemical formulas defined in statements 1-101. These chemical formulas are... Figure 6 Reproduced in [the document]. Specifically, the oligonucleotide moiety represented by Z in any of the following statements may include a nucleic acid for inhibiting SLC25A5 expression as defined in any claim herein.

[0527] 1. A compound comprising the following structure: Formula (I) in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC 1-3Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X1 and X2 are each selected independently from the following group: 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, but with the following condition: (i) q and r cannot both be 0; and (ii) s, t, and v cannot all be 0 at the same time; Z represents the oligonucleotide moiety.

[0528] 2. The compound described in statement 1, wherein R1 is hydrogen each time it appears.

[0529] 3. The compound according to statement 1, wherein R1 is a methyl group.

[0530] 4. The compound according to statement 1, wherein R1 is an ethyl group.

[0531] 5. The compound according to any one of statements 1 to 4, wherein R2 is a hydroxyl group.

[0532] 6. The compound according to any one of statements 1 to 4, wherein R2 is a halogen.

[0533] 7. The compound according to statement 6, wherein R2 is fluorine.

[0534] 8. The compound according to statement 6, wherein R2 is chlorine.

[0535] 9. The compound according to statement 6, wherein R2 is bromine.

[0536] 10. The compound according to statement 6, wherein R2 is iodine.

[0537] 11. The compound according to statement 6, wherein R2 is a nitro group.

[0538] 12. The compound according to any one of statements 1 to 11, wherein X1 is a methylene group.

[0539] 13. The compound according to any one of statements 1 to 11, wherein X1 is oxygen.

[0540] 14. The compound according to any one of statements 1 to 11, wherein X1 is sulfur.

[0541] 15. The compound according to any one of statements 1 to 14, wherein X2 is a methylene group.

[0542] 16. The compound according to any one of statements 1 to 15, wherein X2 is oxygen.

[0543] 17. The compound according to any one of statements 1 to 16, wherein X2 is sulfur.

[0544] 18. The compound according to any one of statements 1 to 17, wherein m = 3.

[0545] 19. The compound according to any one of statements 1 to 18, wherein n = 6.

[0546] 20. The compound according to statements 13 and 15, wherein X1 is oxygen and X2 is methylene, and preferably wherein: q=1, r=2, s=1, t=1, v=1.

[0547] 21. The compound according to statements 12 and 15, wherein X1 and X2 are both methylene groups, and preferably wherein: q=1, r=3, s=1, t=1, v=1.

[0548] 22. A compound according to any one of statements 1 to 21, wherein Z is: in: Z1, Z2, Z3, and Z4 each appear independently as either oxygen or sulfur; One of the bonds between P and Z2 and between P and Z3 is a single bond, and the other is a double bond.

[0549] 23. The compound according to statement 22, wherein the oligonucleotide is an RNA compound capable of regulating (preferably inhibiting) the expression of a target gene.

[0550] 24. The compound according to statement 23, wherein the RNA compound comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, and wherein the first strand and the second strand each have a 5' end and a 3' end.

[0551] 25. The compound according to statement 24, wherein the RNA compound is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0552] 26. The compound according to statement 24, wherein the RNA compound is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0553] 27. A compound of formula (II): Equation (II) 28. A compound of formula (III): Equation (III) 29. The compound according to statement 27 or 28, wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0554] 30. A composition comprising a compound of formula (II) as defined in statement 27 and a compound of formula (III) as defined in statement 28 (optionally subordinate to statement 29).

[0555] 31. The composition according to statement 30, wherein the amount of the compound of formula (III) as defined in statement 28 is 10% to 15% by weight of the composition.

[0556] 32. A compound of formula (IV): Formula (IV) 33. A compound of formula (V): Formula (V) 34. The compound according to statement 32 or 33, wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0557] 35. A composition comprising a compound of formula (IV) as defined in statement 32 and a compound of formula (V) as defined in statement 33 (optionally subordinate to statement 34).

[0558] 36. The composition according to statement 35, wherein the amount of the compound of formula (V) as defined in statement 33 is 10% to 15% by weight of the composition.

[0559] 37. The compound according to any one of statements 1 to 29 or 32 to 34, wherein the oligonucleotide comprises an RNA duplex, the RNA duplex further comprising one or more ribose modified at the 2' position, preferably multiple ribose modified at the 2' position.

[0560] 38. The compound according to statement 37, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluorine, and 2'-deoxy.

[0561] 39. The compound according to any one of statements 1 to 29, 32 to 34, or 37 to 38, wherein the oligonucleotide further comprises one or more degradation-protecting moieties at one or more ends.

[0562] 40. The compound according to statement 39, wherein the one or more degradation protecting moieties are not present at the end of the oligonucleotide chain carrying the ligand moieties, and / or wherein the one or more degradation protecting moieties are selected from thiophosphate nucleoside inter-linked bonds, dithiophosphate nucleoside inter-linked bonds, and reverse abasic nucleosides, wherein the reverse abasic nucleosides are at the distal end of the chain carrying the ligand moieties.

[0563] 41. The compound according to any one of statements 1 to 29, 32 to 34 or 37 to 40, wherein the ligand portion represented by formula (I) in statement 1 comprises one or more ligands.

[0564] 42. The compound according to statement 41, wherein the ligand portion represented by formula (I) in statement 1 comprises one or more carbohydrate ligands.

[0565] 43. The compound according to statement 42, wherein the one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.

[0566] 44. The compound according to statement 43, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0567] 45. The compound according to statement 44, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[0568] 46. ​​The compound according to statement 45, comprising two or three N-acetylgalactosamine moieties.

[0569] 47. The compound according to any one of statements 41 to 46, wherein the one or more ligands are linked in a linear or branched configuration.

[0570] 48. The compound according to statement 47, wherein one or more ligands are connected in a biantennary or triantennary branching configuration.

[0571] 49. The compound according to statements 46 to 48, wherein the portion represented by formula (I) in statement 1: It can be any one of formula (VIa), (VIb) or (VIc), preferably formula (VIa): Formula (VIa) in: A I It is hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and b is an integer from 2 to 5; or Formula (VIb) in: A I It is hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and c and d are independent integers from 1 to 6; or Formula (VIc) in: A I It is hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and e is an integer from 2 to 10.

[0572] 50. The compound according to statements 46 to 48, wherein the portion represented by formula (I) in statement 1: For equation (VII): Equation (VII) in: A I It is hydrogen; a is an integer of 2 or 3.

[0573] 51. The compound described in statement 49 or 50, where a = 2.

[0574] 52. The compound described in statement 49 or 50, where a = 3.

[0575] 53. The compound described in statement 49, where b=3.

[0576] 54. A compound of formula (VIII): Formula (VIII) 55. A compound of formula (IX): Formula (IX) 56. The compound according to statement 54 or 55, wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0577] 57. A composition comprising a compound of formula (VIII) as defined in statement 54 and a compound of formula (IX) as defined in statement 55 (optionally subordinate to statement 56).

[0578] 58. The composition according to statement 57, wherein the amount of the compound of formula (IX) as defined in statement 55 is 10% to 15% by weight of the composition.

[0579] 59. A compound of formula (X): Formula (X) 60. A compound of formula (XI): Formula (XI) 61. The compound according to statement 59 or 60, wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, wherein the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0580] 62. A composition comprising a compound of formula (X) as defined in statement 59 and a compound of formula (XI) as defined in statement 60 (optionally subordinate to statement 61).

[0581] 63. The composition according to statement 62, wherein the amount of the compound of formula (XI) as defined in statement 60 is 10 to 15% by weight of the composition.

[0582] 64. The compound according to any one of statements 54 to 63, wherein the oligonucleotide comprises an RNA duplex, the RNA duplex further comprising one or more modified ribose at the 2' position, preferably multiple modified ribose at the 2' position.

[0583] 65. The compound according to statement 64, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluorine, and 2'-deoxy.

[0584] 66. The compound according to any one of statements 54 to 65, wherein the oligonucleotide further comprises one or more degradation-protecting moieties at one or more ends.

[0585] 67. The compound according to statement 66, wherein the one or more degradation protecting moieties are not present at the end of the oligonucleotide chain carrying the ligand moieties, and / or wherein the one or more degradation protecting moieties are selected from thiophosphate nucleoside inter-linked bonds, dithiophosphate nucleoside inter-linked bonds, and reverse abasic nucleosides, wherein the reverse abasic nucleosides are present at the distal end of the chain carrying the ligand moieties, as shown in any one of formulas (VIII), (IX), (X), or (XI) in any one of statements 54, 55, 59, or 60.

[0586] 68. A method for preparing compounds according to any one of statements 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67 and / or compositions according to any one of statements 30, 31, 35, 36, 57, 58, 62, and 63, comprising reacting a compound of formula (XII) with a compound of formula (XIII): Formula (XII) Formula (XIII) in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X1 and X2 are each selected independently from the following group: 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; and (ii) s, t, and v cannot all be 0 at the same time; Z is the oligonucleotide moiety; And, where appropriate, the ligands are deprotected and / or the second chain of the oligonucleotide moiety is annealed.

[0587] 69. The method according to statement 68, wherein the compound of formula (XII) is prepared by reacting the compound of formula (XIV) with the compound of formula (XV): Formula (XIV) Formula (XV) Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X1 and X2 are each selected independently from the following group: methylene, oxygen, and sulfur; q, r, s, t, v are independent integers from 0 to 4, provided that: (i) q and r cannot both be 0; and (ii) s, t, and v cannot all be 0 at the same time; Z represents the oligonucleotide moiety.

[0588] 70. The method according to statement 68, used to prepare a compound according to any one of statements 20, 25, 27, 29, 54, 56, and / or a composition according to any one of statements 30, 31, 57, 58, wherein: Compounds of formula (XII) have formula (XIIa): Formula (XIIa) And the compound of formula (XIII) has formula (XIIIa): Equation (XIIIa) The oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0589] 71. The method according to statement 68, used to prepare a compound according to any one of statements 20, 25, 28, 29, 55, 56, and / or a composition according to any one of statements 30, 31, 57, 58, wherein: Compounds of formula (XII) have formula (XIIb): Formula (XIIb) And the compound of formula (XIII) has formula (XIIIa): Equation (XIIIa) The oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0590] 72. The method according to statement 68, used to prepare a compound according to any one of statements 21, 26, 32, 34, 59, 61, and / or a composition according to any one of statements 35, 36, 62, 63, wherein: Compounds of formula (XII) have formula (XIIc): Formula (XIIc) And the compound of formula (XIII) has formula (XIIIa): Equation (XIIIa) The oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0591] 73. The method according to statement 68, used to prepare a compound according to any one of statements 21, 26, 33, 34, 60, 61, and / or a composition according to any one of statements 35, 36, 62, 63, wherein: Compounds of formula (XII) have formula (XIId): Formula (XIId) And the compound of formula (XIII) has formula (XIIIa): Equation (XIIIa) The oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0592] 74. The method according to any one of statements 70 to 73, wherein: Compounds of formula (XIIIa) have formula (XIIIb): Equation (XIIIb) 75. According to the method described in statement 69 (which is subordinate to statements 70 to 73), where: Compounds of formula (XIV) have formula (XIVa) or formula (XIVb): Formula (XIVa) Formula (XIVb) And compounds of formula (XV) have formula (XVa) or formula (XIVb): Equation (XVa) Formula (XVb) The oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have 5' and 3' ends, and wherein (i) the RNA duplex is linked at the 5' end of its second strand to an adjacent phosphate ester of formula (XVa), or (ii) the RNA duplex is linked at the 3' end of its second strand to an adjacent phosphate ester of formula (XVb).

[0593] 76. A compound of formula (XII): Formula (XII) in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC 1-3Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X1 and X2 are each selected independently from the following group: methylene, oxygen, and sulfur; q, r, s, t, v are independent integers from 0 to 4, provided that: (i) q and r cannot both be 0; and (ii) s, t, and v cannot all be 0 at the same time; Z represents the oligonucleotide moiety.

[0594] 77. A compound of formula (XIIa): Formula (XIIa) 78. A compound of formula (XIIb): Formula (XIIb) 79. A compound of formula (XIIc): Formula (XIIc) 80. A compound of formula (XIId): Formula (XIId) 81. A compound of formula (XIII): Formula (XIII) in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; m is an integer from 1 to 6; n is an integer from 1 to 10.

[0595] 82. A compound of formula (XIIIa): Equation (XIIIa) 83. A compound of formula (XIIIb): Equation (XIIIb) 84. A compound of formula (XIV): Formula (XIV) in: R1 is selected from the group consisting of hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X2 is selected from the following group: methylene, oxygen, and sulfur; 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.

[0596] 85. A compound of formula (XIVa): Formula (XIVa) 86. A compound of formula (XIVb): Formula (XIVb) 87. A compound of formula (XV): Formula (XV) in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; X1 is selected from the following group: methylene, oxygen, and sulfur; q and r are independent integers from 0 to 4, provided that q and r cannot both be 0 at the same time; Z represents the oligonucleotide moiety.

[0597] 88. A compound of formula (XVa): Equation (XVa) 89. A compound of formula (XVb): Formula (XVb) 90. Use of the compound according to any one of statements 76, 81 to 84, 87 in the preparation of the compound according to any one of statements 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or the composition according to any one of statements 30, 31, 35, 36, 57, 58, 62 and 63.

[0598] 91. Use of the compound according to statement 85 in the preparation of any compound according to any one of statements 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or in any one of statements 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2=F.

[0599] 92. Use of the compound according to statement 86 in the preparation of any compound according to any one of statements 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or in any one of statements 30, 31, 35, 36, 57, 58, 62 and 63, wherein R2=OH.

[0600] 93. Use of the compound according to statement 77 in the preparation of the compound according to any one of statements 20, 25, 27, 29, 54, 56, and / or the composition according to any one of statements 30, 31, 57, 58.

[0601] 94. Use of the compound according to statement 78 in the preparation of the compound according to any one of statements 20, 25, 28, 29, 55, 56, and / or the composition according to any one of statements 30, 31, 57, 58.

[0602] 95. Use of the compound according to statement 79 in the preparation of the compound according to any one of statements 21, 26, 32, 34, 59, 61, and / or the composition according to any one of statements 35, 36, 62, 63.

[0603] 96. Use of the compound according to statement 80 in the preparation of the compound according to any one of statements 21, 26, 33, 34, 60, 61, and / or the composition according to any one of statements 35, 36, 62, 63.

[0604] 97. Use of the compound according to statement 88 in the preparation of the compound according to any one of statements 20, 25, 27 to 29, 54 to 56, and / or the composition according to any one of statements 30, 31, 57, 58.

[0605] 98. Use of the compound according to statement 89 in the preparation of the compound according to any one of statements 21, 26, 32 to 34, 59 to 61, and / or the composition according to any one of statements 35, 36, 62, 63.

[0606] 99. A compound or composition obtained by or through the method according to any one of statements 68 to 75.

[0607] 100. A pharmaceutical composition comprising a compound according to any one of statements 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or a composition according to any one of statements 30, 31, 35, 36, 57, 58, 62 and 63, and a pharmaceutically acceptable carrier, diluent or excipient.

[0608] 101. The compound according to any one of statements 1 to 29, 32 to 34, 37 to 56, 59 to 61 and 64 to 67, and / or the composition according to any one of statements 30, 31, 35, 36, 57, 58, 62 and 63, for use in therapy.

[0609] On the other hand, this application is applicable to the compounds, methods, compositions, or uses covered by the clauses numbered 1-56 below, wherein any reference to a chemical formula in the clauses refers to those chemical formulas as defined in clauses 1-56. These chemical formulas are... Figure 7 Reproduced in [the text]. Specifically, the oligonucleotide moiety represented by Z in any of the following clauses may contain a nucleic acid for inhibiting SLC25A5 expression, as defined in any of the appended claims.

[0610] 1. A compound comprising the following structure: Formula(I*) in: r and s are independent integers selected from 1 to 16; and Z represents the oligonucleotide moiety.

[0611] 2. The compound according to Clause 1, wherein s is an integer selected from 4 to 12.

[0612] 3. The compound according to Clause 2, wherein s is 6.

[0613] 4. The compound according to any one of clauses 1 to 3, wherein r is an integer selected from 4 to 14.

[0614] 5. The compound according to Clause 4, wherein r is 6.

[0615] 6. The compound according to Clause 4, wherein r is 12.

[0616] 7. The compounds described in Clause 5 are subordinate to Clause 3.

[0617] 8. The compounds described in Clause 6 are subordinate to Clause 3.

[0618] 9. The compound according to any one of clauses 1 to 8, wherein Z is: in: Z1, Z2, Z3, and Z4 each appear independently as either oxygen or sulfur; and One of the bonds between P and Z2 and the other between P and Z3 is a single bond, and the other is a double bond.

[0619] 10. The compound according to any one of clauses 1 to 9, wherein the oligonucleotide is an RNA compound capable of regulating (preferably inhibiting) the expression of a target gene.

[0620] 11. The compound according to claim 10, wherein the RNA compound comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, and wherein both the first strand and the second strand have a 5' end and a 3' end.

[0621] 12. The compound according to Clause 11, preferably also subject to Clauses 3 and 6, wherein the RNA compound is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0622] 13. The compound according to Clause 11, preferably also subject to Clauses 3 and 5, wherein the RNA compound is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0623] 14. A compound of formula (II*), preferably subject to clause 12: Equation (II*) 15. A compound of formula (III*), preferably subject to clause 13: Equation (III*) 16. The compound according to any one of clauses 1 to 15, wherein the oligonucleotide comprises an RNA duplex, the RNA duplex further comprising one or more modified ribose at the 2' position, preferably multiple modified ribose at the 2' position.

[0624] 17. The compound according to Clause 16, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluorine, and 2'-deoxy.

[0625] 18. The compound according to any one of clauses 1 to 17, wherein the oligonucleotide further comprises one or more degradation-protecting moieties at one or more ends.

[0626] 19. The compound according to Clause 18, wherein the one or more degradation-protecting moieties are not present at the end of the oligonucleotide chain carrying the linker / ligand moieties, and / or wherein the one or more degradation-protecting moieties are selected from thiophosphate nucleoside inter-linked bonds, dithiophosphate nucleoside inter-linked bonds, and reverse abasic nucleosides, wherein the reverse abasic nucleosides are present on the same chain at the distal end opposite the end carrying the linker / ligand moieties.

[0627] 20. The compound according to any one of Clauses 1 to 19, wherein the ligand portion represented by formula (I*) in Clause 1 comprises one or more ligands.

[0628] 21. The compound according to Clause 20, wherein the ligand portion represented by formula (I*) in Clause 1 comprises one or more carbohydrate ligands.

[0629] 22. The compound according to Clause 21, wherein the one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides or polysaccharides.

[0630] 23. The compound according to Clause 22, wherein the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties.

[0631] 24. The compound according to Clause 23, wherein the one or more carbohydrates comprise one or more N-acetylgalactosamine moieties.

[0632] 25. The compound described in Clause 24, comprising two or three N-acetylgalactosamine moieties.

[0633] 26. The compound according to any one of the preceding clauses, wherein the one or more ligands are linked in a linear or branched configuration.

[0634] 27. The compound according to Clause 26, wherein one or more ligands are connected in a biantennary or triantennary branched structure.

[0635] 28. The compounds according to clauses 20 to 27, wherein the portion represented by formula (I*) in clause 1 is: It can be any one of formula (IV*), (V*) or (VI*), preferably formula (IV*): Formula (IV*) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and b is an integer from 2 to 5; or Formula (V*) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and c and d are independent integers from 1 to 6; or Formula (VI*) in: A I It is a hydrogen or a suitable hydroxyl protecting group; a is an integer of 2 or 3; and e is an integer from 2 to 10.

[0636] 29. The compound according to any one of clauses 1 to 28, wherein the portion represented by formula (I*) in clause 1 is: For equation (VII*): Equation (VII*) in: A I It is hydrogen; a is an integer of 2 or 3.

[0637] 30. The compound according to clause 28 or 29, wherein a = 2.

[0638] 31. The compound according to clause 28 or 29, wherein a = 3.

[0639] 32. The compound according to Clause 28, wherein b=3.

[0640] 33. A compound of formula (VIII*): Formula (VIII*) 34. A compound of formula (IX*): Formula (IX*) 35. The compound according to clause 33 or 34, wherein the oligonucleotide comprises an RNA duplex, the RNA duplex further comprising one or more modified ribose at the 2' position, preferably multiple modified ribose at the 2' position.

[0641] 36. The compound according to Clause 35, wherein the modification is selected from 2'-O-methyl, 2'-deoxy-fluorine, and 2'-deoxy.

[0642] 37. The compound according to any one of clauses 33 to 36, wherein the oligonucleotide further comprises one or more degradation-protecting moieties at one or more ends.

[0643] 38. The compound according to Clause 37, wherein the one or more degradation-protecting moieties are at the end of the oligonucleotide chain carrying the linker / ligand moieties, and / or wherein the one or more degradation-protecting moieties are selected from thiophosphate nucleoside inter-linked bonds, dithiophosphate nucleoside inter-linked bonds, and reverse abasic nucleosides, wherein the reverse abasic nucleosides are located on the same chain at the distal end opposite the end carrying the linker / ligand moieties.

[0644] 39. The compound of claim 33, wherein the oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0645] 40. The compound according to claim 34, wherein the oligonucleotide comprises an RNA duplex containing a first strand and a second strand, wherein the first strand is at least partially complementary to the RNA sequence of the target gene, the second strand is at least partially complementary to the first strand, wherein the first strand and the second strand each have a 5' end and a 3' end, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0646] 41. A method for preparing the compound of any one of clauses 1 to 40, comprising reacting a compound of formula (X*) with a compound of formula (XI*): Formula (X*) Formula (XI*) in: r and s are independent integers selected from 1 to 16; and Z is the oligonucleotide moiety; Furthermore, ligand deprotection and / or annealing of the second chain of oligonucleotides are performed as appropriate.

[0647] 43. The method according to Clause 41, used to prepare the compound according to any one of Clauses 6, 8 to 14, 16 to 33 and 35 to 40, wherein: Compounds of formula (X*) have formula (Xa*): Formula (Xa*) And compounds of formula (XI*) have formula (XIa*): Formula (XIa*) The oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 5' end of its second strand.

[0648] 43. The method according to clause 41, used to prepare the compound according to any one of clauses 5, 7, 9 to 13, 15 to 32 and 34 to 40, wherein: Compounds of formula (X*) have formula (Xb*): Formula (Xb*) Furthermore, compounds of formula (XI*) have formula (XIa*): Formula (XIa*) The oligonucleotide comprises an RNA duplex containing 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 the first strand and the second strand each have 5' and 3' ends, and wherein the RNA duplex is linked to an adjacent phosphate ester at the 3' end of its second strand.

[0649] 44. The method according to clause 42 or 43, wherein: Compounds of formula (XIa*) have formula (XIb*): Formula (XIb*).

[0650] 45. A compound of formula (X*): Formula (X*) in: r is an integer independently selected from 1 to 16; and Z represents the oligonucleotide moiety.

[0651] 46. ​​A compound of formula (Xa*): Formula (Xa*) 47. A compound of formula (Xb*): Formula (Xb*) 48. A compound of formula (XI*): Formula (XI*) in: s is an integer independently selected from 1 to 16; and Z represents the oligonucleotide moiety.

[0652] 49. A compound of formula (XIa*): Formula (XIa*).

[0653] 50. A compound of formula (XIb*): Formula (XIb*).

[0654] 51. Use of the compound according to any one of clauses 45 and 48 to 50 in the preparation of the compound according to any one of clauses 1 to 40.

[0655] 52. Use of the compound according to clause 46 in the preparation of the compound according to any one of clauses 6, 8 to 14, 16 to 33 and 35 to 40.

[0656] 53. Use of the compound according to clause 47 in the preparation of the compound according to any one of clauses 5, 7, 9 to 13, 15 to 32 and 34 to 40.

[0657] 54. A compound or composition that can be obtained by or through any of the methods described in any of clauses 41 to 44.

[0658] 55. A pharmaceutical composition comprising a compound according to any one of clauses 1 to 40 and a pharmaceutically acceptable carrier, diluent or excipient.

[0659] 56. The compound according to any one of clauses 1 to 40, used for therapeutic purposes.

[0660] Example This application can be more fully understood by referring to the following embodiments. However, these embodiments should not be construed as limiting the scope of this application. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and those skilled in the art can make various modifications or changes based on them, and such modifications or changes should be included within the spirit and scope of this application and the appended claims.

[0661] Example 1 - Target Identification background All biological functions originate from the cooperative interactions of hundreds of interacting molecules (mainly proteins), which can be considered as the emergent functional outcome of a protein-protein interaction network, where each node in the network is a protein, and each edge connecting proteins can represent a range of possible interaction types, from complex formation to catalytic activation.

[0662] Historically, this function has been represented as a simple linear pathway. However, with the growth of knowledge, it has become clear that pathways are more complex, and the lowest complexity structure capable of fully representing the functional characteristics of biological processes and capturing resilience to perturbations and robustness to random damage to individual components is the network.

[0663] Networks that underpin biological functions may consist of several interacting typical pathways and additional proteins that primarily participate when typical functions are disrupted. Therefore, it is crucial to develop methods that can model this complexity in a meaningful and tractable manner, and to generate target hypotheses that take into account the inherent resistance to denaturation arising from network robustness.

[0664] Oversimplification in biology and the failure to base drug target selection on realistic biological process models contribute to low drug discovery success rates. Furthermore, a lack of rigorous and objective methods for constructing process network models and prioritizing protein targets within these models means that target selection decisions are often made ad hoc based on preferences or evidence unrelated to functional models.

[0665] Identification process and target methods The identification of the target SLC25A5 / ANT2 began with HepNet, a proprietary computational platform for identifying and validating drug targets in hepatocytes. Two independent approaches were used to identify SLC25A5 / ANT2. The first approach utilized omics data in the form of genome-wide association studies (GWAS) meta-analyses to identify targets for non-alcoholic fatty liver disease (NAFLD) from experimental data. The second approach utilized the HepNet knowledge graph, a semantic network used to describe the interactions between entities such as genes, drugs, diseases, and / or biological processes in the context of hepatocytes.

[0666] For GWAS-based methods, meta-analyses require quality control based on sample size, target population, and the quality of statistical analysis. More specifically, a sample size exceeding 1,000 is used as the cutoff value. If the p-value reflects multiple test correction, the SNPs are proceeded. Finally, sex-merged and all ancestral SNPs (continental Europe, UK, others) were selected; SNPs with sex-specific significance were excluded.

[0667] The final GWAS was used to extract SNPs that were strongly associated with the target trait, in this case NAFLD pathophysiology and related metabolic dysfunctions (e.g., NAFLD and cardiometabolic disease or steatosis and NAFLD risk).

[0668] Subsequently, SNPs were mapped to genes using Variant-to-Gene (V2G) pipeline data from Open Targets Genetics (https: / / genetics.opentargets.org / ). More specifically, for each target SNP, the gene (or multiple genes, if the highest score is copper) with the highest V2G pipeline score was selected, along with genes with scores higher than 0.2.

[0669] This generates a set of genes that, using a proprietary algorithm (Algorithm A), can be used as input data to construct a protein-protein interaction network. The resulting network is then subjected to influence analysis (Algorithm B) to identify enriched biological processes within the network. These processes are subsequently manually evaluated by an internal team to determine the biological processes most relevant to NAFLD target identification.

[0670] A protein-protein interaction network was then constructed around the selected processes, and nodes were scored using a proprietary algorithm (Algorithm C) to assess their importance to the network. The nodes with the highest scores in Algorithm C were manually evaluated by an internal team to identify target therapies for NAFLD metabolic diseases.

[0671] For knowledge graph-based approaches, machine learning algorithms are used to predict interactions between genes and diseases based on the structure of the knowledge graph; this task is often referred to as "link prediction." This generates a set of genes that can be used for network-based target identification using the proprietary algorithm AC, as described above. The networks and other output data of these algorithms differ from those generated by GWAS-based analyses and are evaluated by different internal teams.

[0672] Both analyses independently identified ANT2 / SLC25A5, a key node in the biological processes associated with adipogenesis / non-alcoholic fatty liver disease (referred to as supercluster 574 in Algorithm B).

[0673] Therefore, the inventors analyzed these network models using proprietary analytical methods. These methods utilize directed information to capture key “target” attributes, such as whether a protein is an integrator of information, a key channel for information to other parts of the network, an influencer of key proteins, and the degree to which an influencer is influenced by or influences other proteins (based on the absolute and relative quantities and directions of inputs and outputs). Directed information can also infer hierarchical relationships between proteins. Proteins at higher levels and possessing certain attributes may be more favored than proteins with similar attributes. The relative specificity and strength of each attribute relative to others allows the inventors to score and rank proteins based on their target suitability.

[0674] The ability to characterize the properties of these targets through network relationships enables us to determine their selectivity and effect intensity in selected environments, thereby assessing the suitability of each target for a specific indication.

[0675] Subsequently, proprietary analysis techniques were applied to the network model to identify pharmacologically viable targets. Knockdown of these targets significantly impacted the network, thereby significantly affecting the biological function being modeled. The algorithm fully leverages directed information and hierarchical relationships to identify targets with a range of specific attributes that make them suitable siRNA targets. Targets were then further screened based on therapeutic needs, using protein class and hepatocyte-specific properties.

[0676] The above workflow utilizes proprietary data resources and network node metrics to identify the specific uses of the provided targets.

[0677] The results of the network approach are shown in Table 6. Surprisingly, ANT2 was identified as a drug target for NAFLD, along with various other targets previously associated with metabolic disorders, such as NAFLD (APOA5, HMDH, APOC3, NR1H3, MTP, PCSK9, SOAT1, and ABCA1).

[0678] Table 6

[0679] Example 2: Synthesis of chain 1 General experimental conditions: Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. The compounds were visualized by spraying with methanol (MeOH) or ninhydrin in 5% H₂SO₄ using UV light (254 nm), or by heating, following the Stahl method (from Sigma-Aldrich). Rapid chromatography was performed using a Biotage Sfär Silica 10, 25, 50, or 100 g column (Uppsala, Sweden) on a Biotage Isolera One rapid chromatograph equipped with a dual variable UV wavelength detector (200–400 nm).

[0680] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK Scientific.

[0681] HPLC / ESI-MS was performed using a Waters Acquity UPLC Protein BEH C4 column (300 Å, 1.7 µm, 2.1 x 100 mm) at 60 °C on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile (ACN) containing 0.1% formic acid). The gradient of solvent B increased from 5% to 100% over 15 min at a flow rate of 0.4 mL / min. Detector and conditions: Corona overcharged electrosol detector (from ESA). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0682] At room temperature, on a Varian spectrometer at 500 MHz (1H NMR) and 125 MHz ( 13 (C NMR) records 1H and 13 C10 NMR spectra. Chemical shifts are in ppm, with reference solvent residue peaks (CDCl3–1H NMR: δ 7.26 ppm and 1H NMR). 13 CNMR δ: 77.2 ppm; DMSO-d6-1H NMR: δ: 2.50 ppm and 13The C NMR δ is 39.5 ppm. The coupling constant is in Hertz. The signal splitting mode is described as single peak (s), double peak (d), triple peak (t), or multiple peak (m).

[0683] Synthetic route of the coupling building block TriGalNAc _system 1: 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 mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated aqueous solution of NaHCO3 (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the target compound as a yellow oil. The compound was purified by rapid chromatography (gradient elution: 0-10% MeOH in DCM solution, 10 CV). The product obtained was a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM solution)).

[0684] 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 a 3 Å molecular sieve (5 g) was added to the solution. The mixture was stirred at room temperature for 1 h. Then, TMSOTf (0.77 g, 3.49 mmol, 0.5 eq.) was added to the mixture, and the reaction was stirred overnight. The mixture was filtered through the molecular sieve, 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 product was purified by rapid chromatography (gradient elution: 0-3% MeOH in DCM solution, 10 CV) to give the target compound as a pale yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM solution)). MS: C 20 H 32 N4O 11The calculated value was 504.21, and the measured value was 505.4. ¹H NMR (500 MHz, CDCl₃) δ 6.21–6.14 (m, ¹H), 5.30 (dd, J = 3.4, 1.1 Hz, ¹H), 5.04 (dd, J = 11.2, 3.4 Hz, ¹H), 4.76 (d, J = 8.6 Hz, ¹H), 4.23–4.08 (m, 3H), 3.91–3.80 (m, 3H), 3.74–3.59 (m, 9H), 3.49–3.41 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.97 (d, J = 4.2 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).

[0685] 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 under 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 target compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM solution)). This compound was ready for use without further purification. MS: C 20 H 34 N2O 11 The calculated value is 478.2, and the measured value is 479.4.

[0686] 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 DCM / water mixture (40 mL 1:1 v / v), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 eq.) was added under vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 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 solution, 12 CV) to give the target compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane solution)). MS: C 33 H 53 NO 11 The calculated value is 639.3. The measured value is 640.9. 1H 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). 13C NMR (125 MHz, DMSO-d6) δ 170.3 (3xC), 154.5 (C), 137.1 (C), 128.2 (2xCH), 127.7 (CH), 127.6 (2xCH), 79.7 (3xC), 68.4 (3xCH2), 66.8 (3xCH2), 64.9 (C), 58.7 (CH2), 35.8 (3xCH2), 27.7 (9xCH3).

[0687] Preparation of compound 8: Cbz-NH-tris-Boc-ester 7 (0.20 g, 0.39 mmol, 1.0 eq.) was dissolved in CH2Cl2 (1 mL) under argon atmosphere, and trifluoroacetic acid (TFA, 1 mL) was added. 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 TFA salt of the compound (0.183 g, 98%). This compound can be used without further purification. MS: C 21 H 29 NO 11 The calculated value is 471.6, and the measured value is 472.4.

[0688] Preparation of compound 9: CbzNH-tris-COOH 8 (0.72 g, 1.49 mmol, 1.0 eq.) and GalNAc-PEG3-NH25 (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)urea 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 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and washed with a saturated NaHCO3 aqueous solution (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude product was purified by rapid chromatography (gradient elution: 0-5% MeOH in DCM solution, 14 CV). The resulting product was a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM solution)). MS: C 81 H 125 N7O 41The calculated value is 1852.9, and the measured value is 1854.7. ¹H NMR (500 MHz, DMSO-d6) δ 7.90–7.80 (m, 10H), 7.65–7.62 (m, 4H), 7.47–7.43 (m, 3H), 7.38–7.32 (m, 8H), 5.24–5.22 (m, 3H), 5.02–4.97 (m, 4H), 4.60–4.57 (m, 3H), 4.07–3.90 (m, 10H), 3.67–3.36 (m, 70H), 3.23–3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80-1.78 (m, 17H). 13C NMR (125MHz, 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).

[0689] Preparation of Compound 10: Trianthopteran GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) was dissolved in MeOH (15 mL), with 3 drops of acetic acid (AcOH) and Pd / C (30 mg) added. The reaction mixture was degassed under vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The reaction was monitored by mass spectrometry, and the resulting mixture was filtered through a thin diatomaceous earth pad. The solvent was evaporated, and the residue was dried under high vacuum and used for the next step without further purification. The product was a pale yellow oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 The calculated value is 1718.8, and the measured value is 1719.3.

[0690] 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 DMF solution (5 mL) of 3-azido-1-propylamine (1.0 g, 9.9 mmol, 1.0 eq.) was added dropwise to this solution. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL). The organic layer was separated, dried over Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified by rapid chromatography (gradient elution: 0–5% MeOH in DCM solution, 16 CV). The product was a white solid (1.54 g, 43%, rf = 0.71 (5% MeOH in DCM solution)). MS: C 15 H 23 The calculated value of N5O5 is 353.4, and the measured value is 354.3.

[0691] Preparation of TriGalNAc (12): Under argon atmosphere, compound 10 (0.35 g, 0.24 mmol, 1.0 eq.) and compound 11 (0.11 g, 0.31 mmol, 1.5 eq.) of TriGalNAc were dissolved in DCM (5 mL), and triethylamine (0.1 mL, 0.61 mmol, 3.0 eq.) was added. The reaction was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc (100 mL) and washed with water (100 mL). The organic layer was separated and dried over Na2SO4. The solvent was evaporated, and the crude product was purified by rapid chromatography (elution gradient: 0-10% MeOH in DCM solution, 20 CV) to give the target compound as a white, fluffy solid (0.27 g, 67%, rf = 0.5 (10% MeOH in DCM solution)). MS: C 84 H 137 N 11 O 41 The calculated value is 1957.1, and the measured value is 1959.6.

[0692] Coupling of ligand 1 to the siRNA strand: 5' or 3' end monofluorocyclooctene (MFCO) coupling. 5' end MFCO coupling 3' end MFCO coupling General conditions for MFCO coupling: The amine-modified single chain was dissolved at a concentration of 700 OD / mL in 50 mM carbonate / bicarbonate buffer (pH 9.6) / dimethyl sulfoxide (DMSO) (v / v=4:6), and 1 molar equivalent of 35 mM MFCO-C6-NHS ester (Berry & Associates, catalog number LK 4300) in DMF solution was added to this solution. The reaction was carried out at room temperature, and after 1 h, another molar equivalent of MFCO solution was added. The reaction was continued for 1 h and monitored by liquid chromatography / mass spectrometry (LC / MS). To quantitatively consume the starting material, the amount of MFCO NHS ester reagent should be at least 2 molar equivalents in excess relative to the amine-modified oligonucleotide. The reaction mixture was diluted 15-fold with water, filtered through a Sartorius 1.2 µm filter, and then purified by reversed-phase column chromatography (RP HPLC) on an Äkta Pure instrument (GE Healthcare).

[0693] Purification was performed using a Waters XBridge C18 Prep 19 x 50 mm column. Buffer A was 100 mMMTEAAc (pH 7), and buffer B was buffer A containing 95% acetonitrile. The flow rate was 10 mL / min, and the temperature was 60 °C. UV spectra were recorded at 280 nm. A gradient of 0-100% B was used over 60 column volumes.

[0694] The elution fractions containing full-length coupled oligonucleotides were combined and precipitated in a refrigerator with 3 M NaOAc (pH 5.2) and 85% ethanol. The collected precipitate was then dissolved in water. The sample was desalted by size exclusion chromatography and concentrated using a Speed-Vac concentrator to obtain the coupled oligonucleotides with a separation yield of 40-80%.

[0695] 5'-GalNAc-T1 coupling 3'-GalNAc-T1 coupling The general procedure for TriGalNAc conjugation is as follows: MFCO-modified single chains are dissolved in water at a concentration of 2000 OD / mL, and an equivalent of compound 12 (10 mM) in DMF solution is added to this solution. The reaction is carried out at room temperature, and after 3 h, 0.7 molar equivalents of compound 12 solution are added. The reaction is carried out overnight, and the progress is monitored by LCMS. The conjugate is diluted 15-fold in water, filtered through a 1.2 µm filter membrane of a Sartorius instrument, and then purified by RPHPLC on an Äkta Pure instrument (GE Healthcare).

[0696] RP HPLC purification was performed using a Waters XBridge C18 Prep 19 x 50 mm column. Buffer A was 100 mM triethylammonium acetate (pH 7), and buffer B was buffer A containing 95% acetonitrile. The flow rate was 10 mL / min, and the temperature was 60 °C. UV spectra were recorded at 280 nm. A gradient of 0-100% B was used over 60 column volumes.

[0697] The elution fractions containing full-length coupled oligonucleotides were combined and precipitated in a refrigerator with 3 M NaOAc (pH 5.2) and 85% ethanol. The collected precipitate was dissolved in water to give 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.

[0698] On the Äkta Pure (GE Healthcare) instrument, the conjugates were desalted using size exclusion chromatography with Sephadex G25 Fine resin (GE Healthcare) to obtain conjugated oligonucleotides with a separation yield of 50-70%.

[0699] The following scheme further outlines the synthetic route: Option 1: Option 2: Option 3: Option 4: Option 5: Example 3: Double-chain annealing To generate the desired siRNA duplex, the two complementary strands were annealed by mixing equimolar amounts of the two strands in an aqueous solution. The mixture was placed in a 70°C water bath for 5 minutes, then cooled to room temperature over 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[0700] The doublet was analyzed by analytical SEC HPLC using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system on a Superdex™ 75 Increase 5 / 150 GL column (5 x 153–158 mm, Cytiva). The mobile phase was 1x PBS containing 10% acetonitrile. Isocratic gradient elution was performed at a flow rate of 1.5 mL / min for 10 min at room temperature. UV spectra at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[0701] Example 4: Synthesis of chain 2 General experimental conditions: Thin-layer chromatography (TLC) was performed on silica-coated aluminum plates using a 254 nm fluorescent indicator from Macherey-Nagel. The compounds were visualized by UV light (254 nm) or by spraying with 5% H₂SO₄ in methanol (MeOH) or ninhydrin reagent and heating, following the Stahl method (from Sigma-Aldrich). Rapid chromatography was performed using a Biotage Sfär Silica 10, 25, 50, or 100 g column (Uppsala, Sweden) on a Biotage Isolera One rapid chromatograph equipped with a dual variable UV wavelength detector (200–400 nm).

[0702] All moisture-sensitive reactions were carried out under anhydrous conditions using dry glassware, anhydrous solvents, and an argon atmosphere. All commercially available reagents were purchased from Sigma-Aldrich, and solvents were purchased from Carl Roth GmbH + Co. KG. D-galactosamine pentaacetate was purchased from AK Scientific.

[0703] HPLC / ESI-MS was performed at 60 °C using a Waters Acquity UPLC Protein BEH C4 column (300 Å, 1.7 µm, 2.1 x 100 mm) on a Dionex UltiMate 3000 RS UHPLC system and a Thermo Scientific MSQ Plus mass spectrometer. The solvent system consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile (ACN) containing 0.1% formic acid). The gradient of solvent B increased from 5% to 100% over 15 min at a flow rate of 0.4 mL / min. Detector and conditions: Corona overcharged electrosol detector (from ESA). Nebulizer temperature: 25 °C. N2 pressure: 35.1 psi. Filter: Corona.

[0704] At room temperature, on a Varian spectrometer at 500 MHz (1H NMR) and 125 MHz ( 13 (C NMR) records 1H and 13 C10 NMR spectra. Chemical shifts are in ppm, with reference solvent residue peaks (CDCl3–1H NMR: δ 7.26 ppm and 1H NMR). 13 CNMR δ: 77.2 ppm; DMSO-d6-1H NMR: δ: 2.50 ppm and 13 The C NMR δ is 39.5 ppm. The coupling constant is in Hertz. The signal splitting mode is described as single peak (s), double peak (d), triple peak (t), or multiple peak (m).

[0705] Synthetic route of the conjugated building block TriGalNAc _system 2: 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 h. The reaction mixture was diluted with DCM (50 mL) and washed with cold saturated NaHCO3 aqueous solution (100 mL) and water (100 mL). The organic layer was separated, dried over Na2SO4, and concentrated to give the target compound as a yellow oil. This compound was purified by rapid chromatography (gradient elution: 0-10% MeOH in DCM solution, 10 CV) to give the target compound as a colorless oil (2.5 g, 98%, rf = 0.45 (2% MeOH in DCM solution)).

[0706] 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 3 Å molecular sieve (5 g) was added to the solution. The mixture was stirred at room temperature for 1 h. 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, 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 product was purified by rapid chromatography (gradient elution: 0-3% MeOH in DCM solution, 10 CV) to give the target compound as a pale yellow oil (3.10 g, 88%, rf = 0.25 (2% MeOH in DCM solution)). MS: C 20 H 32 N4O 11 The calculated value was 504.21, and the measured value was 505.4. ¹H NMR (500 MHz, CDCl₃) δ 6.21–6.14 (m, ¹H), 5.30 (dd, J = 3.4, 1.1 Hz, ¹H), 5.04 (dd, J = 11.2, 3.4 Hz, ¹H), 4.76 (d, J = 8.6 Hz, ¹H), 4.23–4.08 (m, 3H), 3.91–3.80 (m, 3H), 3.74–3.59 (m, 9H), 3.49–3.41 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.97 (d, J = 4.2 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 170.6 (C), 170.5 (C), 170.4 (C), 170.3 (C), 102.1 (CH), 71.6 (CH), 70.8 (CH), 70.6 (CH), 70.5 (CH), 70.3 (CH2), 69.7 (CH2), 68.5 (CH2), 66.6 (CH2), 61.5 (CH2), 23.1 (CH3), 20.7 (3xCH3).

[0707] 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 under 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 target compound as a colorless oil (0.95 g, quantitative yield, rf = 0.25 (10% MeOH in DCM solution)). This compound was ready for use without further purification. MS: C 20 H 34 N2O 11 The calculated value is 478.2, and the measured value is 479.4.

[0708] Preparation of Compound 7: Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylamine 6 (3.37 g, 6.67 mmol, 1.0 eq.) was dissolved in a mixture of DCM / water (40 mL 1:1 v / v), and Na2CO3 (0.18 g, 1.7 mmol, 0.25 eq.) was added under vigorous stirring. Benzyl chloroformate (2.94 mL, 20.7 mmol, 3.10 eq.) was added dropwise to the previous mixture, and the reaction was stirred at room temperature for 24 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 solution, 12 CV) to give the target compound as a pale yellow oil (3.9 g, 91%, rf = 0.56 (10% EtOAc in cyclohexane solution)). MS: C 33 H 53 NO 11The calculated value was 639.3, and the measured value was 640.9. ¹H 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). ¹³C NMR (125 MHz, DMSO-d6) δ 170.3 (3xC), 154.5 (C), 137.1 (C), 128.2 (2xCH), 127.7 (CH), 127.6 (2xCH), 79.7 (3xC), 68.4 (3xCH₂), 66.8 (3xCH₂), 64.9 (C), 58.7 (CH₂), 35.8 (3xCH2), 27.7 (9xCH3).

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

[0710] 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)urea 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 h. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (100 mL) and washed with a saturated NaHCO3 aqueous solution (100 mL). The organic layer was dried over Na2SO4, the solvent was evaporated, and the crude product was purified by rapid chromatography (gradient elution: 0-5% MeOH in DCM solution, 14 CV). The obtained product was a pale yellow oil (1.2 g, 43%, rf = 0.20 (5% MeOH in DCM solution)). MS: C 81 H 125 N7O 41The calculated value is 1852.9, and the measured value is 1854.7. ¹H NMR (500 MHz, DMSO-d6) δ 7.90–7.80 (m, 10H), 7.65–7.62 (m, 4H), 7.47–7.43 (m, 3H), 7.38–7.32 (m, 8H), 5.24–5.22 (m, 3H), 5.02–4.97 (m, 4H), 4.60–4.57 (m, 3H), 4.07–3.90 (m, 10H), 3.67–3.36 (m, 70H), 3.23–3.07 (m, 25H), 2.18 (s, 10H), 2.00 (s, 13H), 1.89 (s, 11H), 1.80-1.78 (m, 17H). 13C NMR (125 MHz, DMSO-d6) δ 170.1 (C), 169.8 (C), 169.7 (C), 169.4 (C), 169.2 (C), 169.1 (C), 142.7 (C), 126.3 (CH), 123.9 (CH), 118.7 (CH), 109.7 (CH), 100.8 (CH), 70.5 (CH), 69.8 (CH), 69.6 (CH), 69.5 (CH), 69.3 (CH2), 69.0 (CH2), 68.2 (CH2), 67.2 (CH2), 66.7(CH2), 61.4 (CH2), 22.6 (CH2), 22.4 (3xCH3), 20.7 (9xCH3).

[0711] Preparation of Compound 10: Trianthopteran GalNAc compound 9 (0.27 g, 0.14 mmol, 1.0 eq.) was dissolved in MeOH (15 mL), with 3 drops of acetic acid (AcOH) and Pd / C (30 mg) added. The reaction mixture was degassed under vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The reaction was monitored by mass spectrometry, and the resulting mixture was filtered through a thin diatomaceous earth pad. The solvent was evaporated, and the residue was dried under high vacuum and used for the next step without further purification. The product was a pale yellow oil (0.24 g, quantitative yield). MS: C 73 H 119 N7O 39 The calculated value is 1718.8, and the measured value is 1719.3.

[0712] Preparation of compound 14: Under argon atmosphere, compound 10 (0.45 g, 0.26 mmol, 1.0 eq.) of the tritendon GalNAc, HBTU (0.19 g, 0.53 mmol, 2.0 eq.), and DIPEA (0.23 mL, 1.3 mmol, 5.0 eq.) were dissolved in DCM (10 mL). A DCM solution (5 mL) of compound 13 (0.14 g, 0.53 mmol, 2.0 eq.) was added dropwise to this mixture. The reaction was stirred overnight at room temperature. The solvent was removed, and the residue was dissolved in EtOAc (50 mL), washed with water (50 mL), and dried over Na2SO4. The solvent was evaporated, and the crude product was purified by rapid chromatography (gradient elution: 0–5% MeOH in DCM solution, 20 CV). The product was a white, fluffy solid (0.25 g, 48%, rf = 0.4 (10% MeOH in DCM solution)). MS: C 88 H 137 N7O 42 The calculated value is 1965.1, and the measured value is 1965.6.

[0713] Preparation of TriGalNAc (15): TriGalNAc compound 14 (0.31 g, 0.15 mmol, 1.0 eq.) was dissolved in EtOAc (15 mL), and Pd / C (40 mg) was added. The reaction mixture was degassed under vacuum / argon circulation (3x) and hydrogenated overnight under balloon pressure. The progress of the reaction was monitored by mass spectrometry, and the resulting mixture was filtered through a thin diatomaceous earth pad. The solvent was removed under reduced pressure, and the resulting residue was dried overnight under high vacuum. The residue was used for coupling with oligonucleotides without further purification (0.28 g, quantitative yield). MS: C 81 H 131 N7O 42 The calculated value is 1874.9, and the measured value is 1875.3.

[0714] Conjugation of siRNA strand 2 to the 5' or 3' end of TriGalNAc strand 2 (GalNAc-T2). 5'-GalNAc-T2 coupling 3'-GalNAc-T2 coupling Preparation of TriGalNAc-based chain 2 NHS esters: N-hydroxysuccinimide (NHS) (15.3 mg, 133 µmol) and N,N′-diisopropylcarbodiimide (DIC) (19.7 µL, 127 µmol) were added to a DMF (2.1 mL) solution of carboxylic acid chain 2 (compound 15, 227 mg, 121 µmol). The solution was stirred at room temperature for 18 h and could be used for subsequent coupling reactions without purification.

[0715] The general procedure for triGalNAc series 2 coupling is as follows: The amine-modified single chain is dissolved at a concentration of 700 OD / mL in 50 mM carbonate / bicarbonate buffer pH 9.6 / DMSO (v / v = 4:6), and a one molar equivalent of series 2 NHS ester (57 mM) in DMF is added to this solution. The reaction is carried out at room temperature, and after 1 hour, another molar equivalent of NHS ester solution is added. The reaction is continued for 1 h, and the reaction progress is monitored by LCMS. To quantitatively consume the starting material, the amount of NHS ester reagent should be at least 2 molar equivalents in excess relative to the amine-modified oligonucleotide. The reaction mixture is diluted 15-fold with water, filtered through a Sartorius 1.2 µm filter, and then purified by reversed-phase column chromatography (RP HPLC) on an Äkta Pure instrument (GE Healthcare).

[0716] Purification was performed using a Waters XBridge C18 Prep 19 x 50 mm column. Buffer A was 100 mMMTEAAc (pH 7), and buffer B was buffer A containing 95% acetonitrile. The flow rate was 10 mL / min, and the temperature was 60 °C. UV spectra were recorded at 280 nm. A gradient of 0-100% B was used over 60 column volumes.

[0717] The elution fractions containing full-length coupled oligonucleotides were combined and precipitated in a refrigerator with 3 M NaOAc (pH 5.2) and 85% ethanol, then dissolved in water at a concentration of 1000 OD / mL. O-acetate was removed with 20% ammonium hydroxide aqueous solution until completion (monitored by LC-MS).

[0718] On the Äkta Pure (GE Healthcare) instrument, the conjugates were desalted using size exclusion chromatography with Sephadex G25 Fine resin (GE Healthcare) to obtain conjugated oligonucleotides with a separation yield of 60-80%.

[0719] The conjugates were characterized by HPLC-MS analysis using a 2.1 x 50 mm XBridge C18 column (Waters) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system equipped with a Compact ESI-Qq-TOF mass spectrometer (Bruker Daltonics). Buffer A was an aqueous solution containing 16.3 mM triethylamine, 100 mM HFIP, and 1% MeOH, while buffer B was buffer A containing 95% MeOH. The flow rate was 250 µL / min, and the temperature was 60 °C. UV spectra were recorded at 260 and 280 nm using a gradient of 1-100% B over 31 minutes.

[0720] The following scheme further outlines the synthetic route: Option 6: Option 7: Option 8: Option 9: Example 5: Double-chain annealing To generate the desired siRNA duplex, the two complementary strands were annealed by mixing equimolar amounts of the two strands in an aqueous solution. The mixture was placed in a 70°C water bath for 5 minutes, then cooled to room temperature over 2 hours. The duplex was lyophilized for 2 days and stored at -20°C.

[0721] The doublet was analyzed by analytical SEC HPLC using a Dionex Ultimate 3000 (Thermo Fisher Scientific) HPLC system on a Superdex™ 75 Increase 5 / 150 GL column (5 x 153–158 mm, Cytiva). The mobile phase was 1x PBS containing 10% acetonitrile. Isocratic gradient elution was performed at a flow rate of 1.5 mL / min for 10 min at room temperature. UV spectra at 260 and 280 nm were recorded. Water (LC-MS grade) was purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS; 10x, pH 7.4) was purchased from GIBCO (Thermo Fisher Scientific).

[0722] Example 6: Alternative synthetic route for the conjugated structural unit TriGalNAc_system 2 Binding of TriGalNAc lineage 2 to the siRNA strand: Coupling at the 5' or 3' end of TriGalNAc lineage 2 (GalNAc-T2). Coupling conditions Pre-activation: At 25 °C, TFA-O-PFP (15 μl, 21 eq.) was added to a DMF solution (160 μL) of compound 15 (16 μol, 4 eq.), followed by DIPEA (23 μl, 32 eq.). The tube was shaken at 25 °C for 2 h. The reaction was terminated with H2O (10 μL).

[0723] Couplet: The resulting mixture was diluted with DMF (400 μl), and then an oligoamine solution (4.0 μmol, dissolved in 10x PBS, pH 7.4, 500 μL; final concentration of oligonucleotide in organic and aqueous solutions: 4 μmol / ml = 4 mM) was added. The tubes were shaken at 25 °C for 16 h, and the reaction product was analyzed by LCMS. The resulting mixture was treated with 28% NH4OH (4.5 ml) and shaken at 25 °C for 2 h. The mixture was analyzed by LCMS, concentrated, and purified by IP-RP HPLC to obtain oligonucleotides coupled to the 2-GalNAc chain.

[0724] 5'-GalNAc-T2 coupling 3'-GalNAc-T2 coupling Example 7: Solid-phase synthesis method: scale ≤ 1 μmol The synthesis of siRNA sense and antisense strands was performed on a MerMade 192X synthesizer using a commercially available solid-phase support made of glass with controllable pore size and a universal adapter (universal CPG, loading volume 40 μmol / g; LGCBiosearch or Glen Research). RNA phosphoramide was purchased from ChemGenes or Hongene.

[0725] The 2'-O-methylphosphoramides used are as follows: 5'-(4,4'-dimethoxytriphenylmethyl)-N-benzoyladenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetylglysine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-isobutyrylguanosine 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.

[0726] The 2'-F phosphoramids used are as follows: 5'-dimethoxytriphenylmethyl-N-benzoyldeoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-dimethoxytriphenylmethyl-N-acetyldeoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-dimethoxytriphenylmethyl-N-isobutyryldeoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, and 5'-dimethoxytriphenylmethyldeoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid.

[0727] All phosphorusamides were soluble in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyl-uridine phosphorusamide, which was soluble in DMF / MeCN (1:4, v / v). 0.02 M iodine (DNAchem) dissolved in acetonitrile / pyridine / water was used as the oxidizing agent. The sulfidation reaction of the thiophosphate bond was carried out using 0.2 M PADS (TCI) in acetonitrile / pyridine (1:1 v / v). A 0.25 M mM acetonitrile solution of 5-ethylthiotetrazole (ETT) was used as the activating agent.

[0728] Inverted base-free phosphoramidite, 3-O-dimethoxytriphenylmethyl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[0729] In each cycle, DMT was removed with a deprotection solution (3% TCA in DCM solution, DNAchem).

[0730] The coupling time is 180 seconds. The oxidant contact time is set to 80 seconds, and the vulcanization time is 2 * 100 seconds.

[0731] At the end of the synthesis, the oligonucleotides were cleaved from the solid support by treatment with an NH4OH:EtOH solution (volume ratio 4:1) at 45°C (TCI) for 20 hours. The solid support was then filtered off, the filter membrane was thoroughly washed with water, and the volume of the combined solutions was reduced by vacuum evaporation.

[0732] Oligonucleotides were treated with ultracentrifugation to form sodium salts using an Amicon Ultra-2 centrifuge filter and PBS buffer (10x, Teknova, pH 7.4, sterile), or with EtOH precipitation of 1M sodium acetate to form sodium salts.

[0733] Single-strand identity was assessed by MS ESI, followed by annealing in water to form the final double-stranded siRNA, and double-strand purity was assessed by size exclusion chromatography.

[0734] Example 8: Solid-phase synthesis method: scale ≥ 5 µmol The sense and antisense strands of siRNA were synthesized at a scale of 5 µmol on a MerMade 12 synthesizer using a commercially available solid-phase support made of glass with controlled pore size and a universal adapter (universal CPG, loading volume 40 μmol / g; LGC Biosearch or Glen Research). 12 µmol of the sense strand for 3' coupling was synthesized on a 500 Å solid-phase support containing the 3'-PT-amino modifier C6 CPG at a loading volume of 86 µmol / g (LGC).

[0735] RNA phosphoramide was purchased from ChemGenes or Hongene.

[0736] The 2'-O-methylphosphoramides used are as follows: 5'-(4,4'-dimethoxytriphenylmethyl)-N-benzoyladenosine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-acetylglysine 2'-O-methyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, 5'-(4,4'-dimethoxytriphenylmethyl)-N-isobutyrylguanosine 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.

[0737] The 2'-F phosphoramids used are as follows: 5'-dimethoxytriphenylmethyl-N-benzoyldeoxyadenosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-dimethoxytriphenylmethyl-N-acetyldeoxycytidine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, 5'-dimethoxytriphenylmethyl-N-isobutyryldeoxyguanosine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid, and 5'-dimethoxytriphenylmethyldeoxyuridine 2'-fluoro-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramid.

[0738] Inverted base-free phosphoramidite, 3-O-dimethoxytriphenylmethyl-2-deoxyribose-5-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, purchased from Chemgenes (ANP-1422) or Hongene (OP-040).

[0739] Except for 2'-O-methyluridine phosphoramidite, which was soluble in DMF / MeCN (1:4, v / v), all phosphoramidites were soluble in anhydrous acetonitrile (Honeywell Research Chemicals) at a concentration of 0.05 M, except for 2'-O-methyluridine phosphoramidite, which was soluble in DMF / MeCN (1:4, v / v). 0.02 M iodine (DNAchem) dissolved in acetonitrile / pyridine / water was used as the oxidizing agent. The sulfidation reaction of the thiophosphate bond was carried out using 0.2 M PADS (TCI) in acetonitrile / pyridine (1:1 v / v). A 0.25 M mM acetonitrile solution of 5-ethylthiotetrazole (ETT) was used as the activating agent.

[0740] In each cycle, DMT was removed with a deprotection solution (3% TCA dissolved in DCM, DNAchem).

[0741] For chains synthesized on a general-purpose CPG, coupling was performed using 8 equivalents of amidite over a reaction time of 130 seconds. Oxi...

Claims

1. An inhibitor of SLC25A5 / ANT2 expression and / or function, wherein the inhibitor is coupled to one or more ligand moieties, preferably wherein the ligand moieties allow targeting of hepatocytes.

2. The inhibitor according to claim 1, wherein the inhibitor is a siRNA oligomer.

3. An inhibitor of SLC25A5 / ANT2 expression and / or function, wherein the inhibitor is a siRNA oligomer.

4. The inhibitor of claim 3, wherein the inhibitor comprises an siRNA oligomer coupled to one or more ligand moieties, preferably wherein the ligand moieties allow targeting of hepatocytes.

5. The inhibitor according to claim 1, 2 or 4, wherein the one or more ligand portions comprise one or more GalNAc ligands or comprise one or more GalNAc ligand derivatives.

6. The inhibitor according to claim 1, 2 or 4, wherein the one or more ligand portions comprise one or more GalNAc ligand derivatives.

7. The inhibitor according to one or more of the preceding claims, wherein the target of the inhibitor is SLC25A5 / ANT2.

8. The inhibitor according to one or more of the preceding claims, wherein the inhibitor is a nucleic acid for inhibiting SLC25A5 expression, the nucleic acid comprising a double-stranded segment, the double-stranded segment comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) At least partially complementary to the RNA portion transcribed from the SLC25A5 gene, and (ii) Contains at least 17 consecutive nucleosides that differ from SEQ ID NO: 280 or SEQ ID NO: 304 by 0 or 1 nucleoside.

9. The inhibitor according to one or more of the preceding claims, wherein the inhibitor is a nucleic acid for inhibiting SLC25A5 expression, the nucleic acid comprising a double-stranded segment, the double-stranded segment comprising a first strand and a second strand at least partially complementary to the first strand, wherein the first strand: (i) At least partially complementary to the RNA portion transcribed from the SLC25A5 gene, and (ii) Contains at least 17 consecutive nucleosides that differ from SEQ ID NO: 832 or SEQ ID NO: 856 by 0 or 1 nucleoside.

10. The inhibitor according to claim 8 or 9, wherein the first chain comprises nucleoside 2-18 of any sequence defined in claim 8 or 9.

11. The inhibitor of claim 8, wherein the second chain comprises a nucleoside sequence of at least 17 consecutive nucleosides differing from SEQ ID NO: 556 or SEQ ID NO: 580 by 0 or 1 nucleoside, and wherein the second chain has a region on the 17 consecutive nucleosides that is at least 85% complementary to the first chain.

12. The inhibitor of claim 9, wherein the second chain comprises a nucleoside sequence of at least 17 consecutive nucleosides differing from SEQ ID NO: 1108 or SEQ ID NO: 1132 by 0 or 1 nucleoside, and wherein the second chain has a region on the 17 consecutive nucleosides that is at least 85% complementary to the first chain.

13. The inhibitor of claim 8, wherein the first chain comprises SEQ ID NO: 280 or SEQ ID NO:

304.

14. The inhibitor of claim 9, wherein the first chain comprises SEQ ID NO: 832 or SEQ ID NO:

856.

15. The inhibitor of claim 11, wherein the second chain comprises SEQ ID NO: 556 or SEQ ID NO:

580.

16. The inhibitor of claim 12, wherein the second chain comprises SEQ ID NO: 1108 or SEQ ID NO: 1132.

17. The inhibitor according to any one of claims 8 and 11, comprising a first chain and a second chain, wherein the first chain and the second chain comprise a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside, consists of a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside, or consists substantially of a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside: 。 18. The inhibitor according to any one of claims 9 and 12, comprising a first chain and a second chain, the first chain and the second chain comprising a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside, consisting of a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside, or substantially consisting of a nucleoside sequence differing from any of the following combinations of the first sequence and the second sequence by 0 or 1 nucleoside: 。 19. The inhibitor according to any one of claims 8 to 18, wherein the length of the first chain is 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19 or 23 nucleosides.

20. The inhibitor according to any one of claims 8 to 19, wherein the length of the second chain is 17 to 30 nucleosides, preferably 19 to 25 nucleosides, more preferably 19, 21, or 23 nucleosides.

21. The inhibitor according to any one of claims 8 to 20, wherein the length of the double-stranded segment of the nucleic acid is 17 to 30 nucleotides, more preferably 19, 21 or 23 nucleotides.

22. The inhibitor according to any one of claims 8 to 21, wherein the length of the complementary region between the first strand and the RNA portion transcribed from the SLC25A5 gene is 17 to 30 nucleotides.

23. The inhibitor according to any one of claims 8 to 22, wherein the nucleic acid further comprises one or more single-stranded nucleoside overhangs, optionally wherein the overhangs are present on the first or second strand, preferably at the 3' end of the first or second strand, and / or wherein the overhangs comprise 1 to 4 nucleosides, more preferably 2 nucleosides.

24. The inhibitor according to any one of claims 8 to 23, wherein the nucleic acid is an siRNA oligonucleotide.

25. The inhibitor according to any one of claims 8 to 24, wherein the second sense chain further comprises one or more abasic nucleosides in the terminal region of the second chain, and wherein the abasic nucleosides are linked to adjacent nucleosides via reverse internucleotide bonds.

26. The inhibitor according to any one of claims 8 to 25, wherein the second chain comprises two consecutive abasic nucleosides in its 5' terminal region, one of such abasic nucleosides being a terminal nucleoside in the 5' terminal region of the second chain, and the other abasic nucleoside being the penultimate nucleoside in the 5' terminal region of the second chain, wherein: (a) The penultimate abasic nucleotide is connected to the adjacent first basic nucleotide in the adjacent 5' proximal region via a reverse internucleotide bond; and (b) The reverse key is a 5-5' reverse key; and (c) When read toward the end containing the terminal and the penultimate abase-free nucleotide, the bond between the terminal and the penultimate abase-free nucleotide is 3'5'.

27. The inhibitor of claim 25 or 26, or the inhibitor for the purpose thereof, wherein the reverse nucleoside internucleotide bond is located at the distal end of the 5' end region of the second chain, or at the distal end of the 3' end region of the second chain.

28. The inhibitor according to claim 26, wherein (i) The first chain and the second chain each have a length of 23 nucleotides; (ii) The two thiophosphate nucleoside bonds are located between three consecutive positions in the 5' proximal terminal region of the second chain, wherein, The first thiophosphate nucleoside inter-bond exists between the adjacent first basic nucleoside of (a) and the adjacent second basic nucleoside in the 5' proximal region of the second chain, and the second thiophosphate nucleoside inter-bond exists between the adjacent second basic nucleoside and the adjacent third basic nucleoside in the 5' proximal region of the second chain. (iii) Two thiophosphate nucleoside bonds are located between three consecutive positions in the 5' and 3' terminal regions of the first chain, respectively, thereby connecting the terminal nucleosides at the 5' and 3' terminal regions of the first chain to the corresponding penultimate 5' and 3' nucleosides via thiophosphate nucleoside bonds, and each first penultimate 5' and 3' nucleoside is connected to the corresponding penultimate 5' and 3' nucleoside via thiophosphate nucleoside bonds; and (iv) The second strand of the nucleic acid is directly or indirectly coupled to one or more ligand moieties in the 3' end region of the second strand.

29. The inhibitor according to claim 26 or 28, wherein the two consecutive inverted anucleotides in the 5' terminal region of the second chain are present as the following 5' terminal motif: in: T represents 2'Me ribose modification. B represents the nucleoside bases of the first two basic nucleosides in the 5' terminal region of the second chain. Z represents the remaining 19 consecutive basic nucleosides in the second chain.

30. The inhibitor according to any one of claims 8 to 29, wherein the nucleic acid is directly or indirectly coupled to one or more ligand moieties, optionally wherein the ligand moieties are present in the terminal region of the second strand, preferably in the 3' terminal region of the second strand.

31. The inhibitor of claim 30, wherein the ligand portion comprises: (i) One or more N-acetylgalactosamine (GalNAc) ligands, and / or (ii) One or more N-acetylgalactosamine (GalNAc) ligand derivatives.

32. The inhibitor of claim 31, wherein one or more GalNAc ligands and / or GalNAc ligand derivatives are directly or indirectly coupled to the 5' or 3' terminal region of the second strand of the nucleic acid, preferably coupled to its 3' terminal region.

33. The inhibitor according to any one of claims 30 to 32, comprising the following structure: in: Each time R1 appears, it is independently selected from the following group: hydrogen, methyl, and ethyl; R2 is selected from the following group: hydrogen, hydroxyl group, -OC 1-3 Alkyl group, -C(=O)OC 1-3 Alkyl, halogen, and nitro groups; X1 and X2 are each selected independently from the following group: 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; and (ii) s, t, and v cannot all be 0 at the same time; Z is the oligonucleotide moiety.

34. The inhibitor according to claim 33, comprising the following structure The oligonucleotides therein represent consecutive nucleosides of the second chain.

35. The inhibitor according to any one of claims 30 to 32, comprising the following structure: in: r and s are independent integers selected from 1 to 16; and Z represents the oligonucleotide moiety.

36. The inhibitor according to claim 35, comprising the following structure Oligonucleotides represent consecutive nucleosides of the second chain.

37. The inhibitor according to any one of claims 34 or 36, wherein the structure is coupled to the 3' end region of the second chain.

38. The inhibitor according to any one of claims 18, 29, 34 and 37.

39. The inhibitor according to any one of claims 18, 29, 36 and 37.

40. The inhibitor according to one or more of the preceding claims, which is formulated together with an excipient and / or a carrier into a pharmaceutical composition.

41. A pharmaceutical composition comprising a combination of an inhibitor as described in one or more of the preceding claims and a pharmaceutically acceptable excipient or carrier.

42. The inhibitor according to any one of claims 1 to 40 or the pharmaceutical composition according to claim 41, for use in therapy.

43. The inhibitor according to any one of claims 1 to 40 or the pharmaceutical composition according to claim 41, for the prevention and / or treatment of metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity, and / or diseases or conditions associated with lipogenesis, and / or for the reduction of lipogenesis.

44. Use of SLC25A5 / ANT2 as a target for identifying one or more therapeutic agents for the prevention and / or treatment of metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity and / or diseases or conditions associated with lipogenesis, and / or for reducing lipogenesis.

45. A method for treating or preventing metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD), and / or obesity and / or diseases or conditions associated with lipogenesis, and / or a method for reducing lipogenesis, comprising: Administering an SLC25A5 / ANT2 inhibitor to a patient, such as an inhibitor as defined in any one of claims 1 to 40, or a pharmaceutical composition comprising an SLC25A5 / ANT2 inhibitor, such as a composition as defined in claim 41.

46. ​​The use of the inhibitor of any one of claims 1 to 40 or the pharmaceutical composition of claim 41 in the preparation of a medicament for treating metabolic diseases or conditions such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis, and / or for reducing lipogenesis.

47. Use of SLC25A5 / ANT2 as a biomarker for metabolic diseases or conditions such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis.

48. An in vivo method for predicting susceptibility to metabolic diseases or conditions, such as those associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or lipogenesis-related diseases or conditions, typically by monitoring the sequence and / or expression level and / or function of SLC25A5 / ANT2 in samples obtained from patients.

49. A method for predicting a patient's susceptibility to metabolic diseases or conditions, such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with lipogenesis, and optionally treating said metabolic diseases or conditions, the method comprising: (a) Obtaining samples from patients (b) Detect the sequence and / or expression and / or function of SLC25A5 / ANT2 in the samples obtained from the patient. (c) Based on the sequence and / or expression and / or function of SLC25A5 / ANT2 in the samples obtained from the patients, predict susceptibility to metabolic diseases or conditions such as metabolic diseases or conditions associated with non-alcoholic fatty liver disease (NAFLD) and / or obesity and / or diseases or conditions associated with adipogenesis. (d) Preferably, an effective amount of an SLC25A5 / ANT2 inhibitor is administered to a confirmed patient, preferably the SLC25A5 / ANT2 inhibitor according to any one of claims 1 to 40, or a pharmaceutical composition containing an SLC25A5 / ANT2 inhibitor, such as the composition defined in claim 41.

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