Short antisense nucleotide targeting proximal renal tubule

Abstract

The present invention relates to the fields of nucleic acid drugs and pharmaceutical chemistry, and in particular to a short antisense nucleotide targeting a proximal renal tubule. Provided is an antisense oligonucleotide targeting a proximal tubular cell, or a pharmaceutically acceptable salt thereof. The antisense oligonucleotide has a structure comprising a 5'-wing region, a spacer region, and a 3'-wing region, wherein the 5'-wing region consists of 1-5 nucleotides having a first chemical modification, and the 3'-wing region consists of 1-5 nucleotides having a second chemical modification.

Description

Short antisense nucleotides targeting the proximal renal tubules

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. 2024117383870, filed on November 29, 2024; Chinese patent application No. 2025106166507, filed on May 14, 2025; and Chinese patent application No. 2025111440041, filed on August 15, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the fields of nucleic acid drugs and medicinal chemistry, specifically to short antisense nucleotides that target the proximal renal tubules. Background Technology

[0004] Nucleic acid drugs selectively regulate gene expression through the engineering of nucleotide sequences. Common nucleic acid drugs include antisense oligonucleotides (ASO), aptamers, short interfering RNA (siRNA), and microRNA (miRNA). These drugs differ from traditional drugs in their mechanism of action; they can be precisely controlled by altering their sequence, structure, or chemical modifications, and their flexibility allows for tailoring drugs to specific disease needs.

[0005] Compared to traditional small molecule and protein drugs, nucleic acid drugs have unique advantages. Fundamentally, the main targets of traditional small molecule drugs and antibody drugs are proteins, but only 1.5% of the human genome encodes proteins, and 80% of these are untreatable targets for traditional drugs. Unlike traditional drugs, nucleic acid drugs regulate target expression by binding to relevant nucleic acids, meaning that nucleic acid drugs are theoretically applicable to any therapeutic target. Furthermore, once the base sequence of the target gene is known, designing nucleic acid drugs that bind to the target gene is relatively easy. Finally, due to the simplicity and low cost of nucleotide synthesis, the development of nucleic acid drugs is quite readily available.

[0006] Antisense oligonucleotides (ASOs) are short, single-stranded nucleic acid sequences that regulate gene expression or inhibit protein translation by binding complementary to target RNA molecules. Two main mechanisms by which ASOs exert their function have been identified: RNase H1-dependent cleavage (RNA cleavage) and RNase H1-independent mechanisms (steric hindrance). In the RNase H1-dependent mechanism, after binding to the target RNA (nuclear / cytoplasmic), RNase H1 cleaves the target RNA strand (7-10 nt from the 5' end of the double-stranded region). In the RNase H1-independent mechanism, high-affinity binding generates steric hindrance, modifying gene expression without degrading RNA. ASOs can also alter the biological properties of proteins by binding to pre-mRNA, hiding cleavage signals.

[0007] Gapmer technology represents the second generation of oligonucleotide drugs. Gapmer technology involves introducing chemical modifications such as LNA, 2'-OMe, or 2'-F into oligonucleotide molecules. The goal is to increase the binding of small nucleic acids to target sequences, enhance nuclease stability, optimize pharmacokinetic properties, and minimize side effects. It is crucial for the transformation of oligonucleotide drugs into therapeutic agents.

[0008] Currently, the vast majority of ASO drugs approved by the FDA or in clinical trials are 16-20 megahertz in length. In the development of ASO drugs targeting the kidneys, antisense nucleic acids in gap-mer form (16 megahertz or longer) are commonly used. Summary of the Invention

[0009] This disclosure, through the design of Gapmer ASOs of different lengths and with different modifications, has shown that Gapmer ASOs of 12-mer length and / or with LNA and cEt modifications have the best inhibitory effect on target gene expression, especially in proximal renal tubular cells.

[0010] Specifically, this disclosure provides the following technical solutions:

[0011] On one hand, the present invention provides an antisense oligonucleotide or a pharmaceutically acceptable salt thereof that targets proximal renal tubular cells. The structure of the antisense oligonucleotide is a 5'-wing region-spacer region-3'-wing region, wherein the 5'-wing region consists of 1-5 nucleotides with a first chemical modification, and the 3'-wing region consists of 1-5 nucleotides with a second chemical modification. The 1-5 nucleotides include 1, 2, 3, 4, or 5.

[0012] Preferably, the antisense oligonucleotide is 10-18 nucleotides in length, that is, the antisense oligonucleotide is composed of 10-18 nucleotides, specifically including 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0013] Preferably, the antisense oligonucleotide is 12 or 13 nucleotides in length, that is, the antisense oligonucleotide is composed of 12 or 13 nucleotides.

[0014] Preferably, the 5' wing region consists of 2 or 3 nucleotides with a first chemical modification.

[0015] Preferably, the 3' wing region consists of two or three nucleotides with a second chemical modification.

[0016] Preferably, the antisense oligonucleotide has a 2-8-2 or 2-8-3 modification pattern, wherein the first number represents the number of nucleotides in the 5' wing region, the second number represents the number of nucleotides in the spacer region, and the third number represents the number of nucleotides in the 3' wing region.

[0017] Preferably, the first chemical modification involves attaching one or more first linking groups between the 4' and 2' positions of the ribose.

[0018] Preferably, the second chemical modification involves attaching one or more second linking groups between the 4' and 2' positions of the ribose.

[0019] Preferably, the first linking group and the second linking group each independently comprise -[C(R1)(R2)] n -, -C(R1)=C(R2)-, -C(R1)=N-, -C(=NR1)-, -C(=O)-, -C(=S)-, -O-, -Si(R1)2-, -S(=O) x -、-N(R1)-、-R-[C(R1)(R2)]n-R'-、-[C(R1)(R2)]nR-R'- or -R-R'-[C(R1)(R2)]n-、-RC(R1)=C(R2)-R'-、-R-R'-C(R1)=C(R2)-、-C(R1)=C(R2)-R-R'-;wherein, R and R', each independently, include single bonds, -N(R3)-、-O-、-S-、-Se-、-Si(R4)(R5)-、-C(=O)-、-C(=S)-、-C(=NR3)-;

[0020] Where x is 0, 1, or 2;

[0021] n is 1, 2, 3 or 4;

[0022] R1, R2, R3, R4, and R5, each independently, include H, a protecting group, a hydroxyl group, and substituted or unsubstituted C1-C. 12 Alkyl, substituted or unsubstituted C 2- C 12 alkenyl, substituted or unsubstituted C2-C12 Alkyne, substituted or unsubstituted C5-C 20 Aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroaryl, substituted or unsubstituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-J1), substituted acyl, CN, sulfonyl (S(=O)2-J1), sulfonyloxy (S(=O)-J1) or -C(=NH2)-NH2, or R1 and R2 linked together to form C3-C6 cycloalkyl or heterocyclic alkyl;

[0023] J1 and J2, each independently, include H, substituted or unsubstituted C1-C. 12 Alkyl, substituted or unsubstituted C2-C 12 alkenyl, substituted or unsubstituted C2-C 12 Alkyne, substituted or unsubstituted C5-C 20 Aryl, substituted or unsubstituted acyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted C1-C 12 Aminoalkyl or protecting group;

[0024] Preferably, the first linking group and the second linking group each independently include -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -[C(R1)(R2)]nN(R3)-, [C(R1)(R2)]nN(R3)-O-, -[C(R1)(R2)]nON(R3)-, -[C(R1)(R2)]nS-, -C(R1)=C(R2)-, -C(R1)=N-; wherein R1, R2, and R3 each independently are H, a protecting group, or C1-C 12 alkyl;

[0025] Preferably, the first and second linking groups each independently include -(CH2)3-, -(CH2)2-, -CH2-O-, -(CH2)2-O-, -CH2-ON(R1)-, -CH2-N(R1)-O-, -CH(CH3)-O-, -C(C2H4)O-, -CH2-N(R1)-, -CH(R1)-NH-, -CH(R1)-N(R1)-, CH2-S-, -CH(CH3)-S-, -CH2-R1-, or -C(=O)-N(R1)-, wherein each R1 is independently H, a protecting group, or C1-C. 12 alkyl;

[0026] Preferably, the first linking group and the second linking group are each independently -CH2-O- or -CH(CH3)-O-.

[0027] Preferably, the first linking group and the second linking group are the same or different, and the first chemical modification is the same or different from the second chemical modification.

[0028] Preferably, the first linking group and the second linking group are the same, and the first chemical modification and the second chemical modification are the same.

[0029] In one specific embodiment, the first linking group is -CH2-O-, and the second linking group is -CH2-O-.

[0030] In one specific implementation, the first linking group is -CH(CH3)-O-, and the second linking group is -CH(CH3)-O-.

[0031] In one specific implementation, the first chemical modification is the attachment of -CH2-O- between the 4' and 2' positions of the ribose.

[0032] In one specific embodiment, the second chemical modification is the attachment of -CH2-O- between the 4' and 2' positions of the ribose.

[0033] In one specific implementation, the first chemical modification is the attachment of -CH(CH3)-O- between the 4' and 2' positions of the ribose.

[0034] In one specific embodiment, the second chemical modification is the attachment of -CH(CH3)-O- between the 4' and 2' positions of the ribose.

[0035] Preferably, when the first or second linker is -CH2-O-, the nucleotide connecting the first or second linker between the 4' and 2' positions of the ribose is an LNA nucleotide, which has the following structure:

[0036] Where Bx refers to a base.

[0037] Preferably, when the first or second linker is -CH(CH3)-O-, the nucleotide linking the first or second linker between the 4' and 2' positions of the ribose is a restricted ethyl-modified nucleotide having the following structure:

[0038] Where Bx refers to a base.

[0039] In some embodiments, the LNA nucleotide may be in the α-L configuration or the β-D configuration. When a particular bicyclic nucleotide (e.g., LNA or cEt) is specified in the examples herein, it may be in the α-L configuration or the β-D configuration unless otherwise stated.

[0040] In one specific embodiment, the structure of the antisense oligonucleotide is a 5' wing region-spacer region-3' wing region. The antisense oligonucleotide has a 2-8-2 or 2-8-3 modification pattern, wherein the 5' wing region consists of two nucleotides with a first chemical modification, the 3' wing region consists of two to three nucleotides with a second chemical modification, and the spacer region consists of eight modified or unmodified nucleotides. The first chemical modification is the connection of -CH2-O- between the 4' and 2' positions of the ribose, and the second chemical modification is the connection of -CH2-O- between the 4' and 2' positions of the ribose.

[0041] In one specific embodiment, the structure of the antisense oligonucleotide is a 5' wing region-spacer region-3' wing region. The antisense oligonucleotide has a 2-8-2 or 2-8-3 modification pattern, wherein the 5' wing region consists of two nucleotides with a first chemical modification, the 3' wing region consists of two to three nucleotides with a second chemical modification, and the spacer region consists of eight modified or unmodified nucleotides. The first chemical modification is the connection of -CH(CH3)-O- between the 4' and 2' positions of the ribose, and the second chemical modification is the connection of -CH(CH3)-O- between the 4' and 2' positions of the ribose.

[0042] Preferably, the antisense oligonucleotide also has chemical modifications that are not the first chemical modification or the second chemical modification, specifically, such as base modification, ribose modification and / or phosphate backbone modification.

[0043] In some implementations, the modified adenine has structure (I):

[0044] Among them, R 2A H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 thioalkyl, or substituted C1-C6 thioalkyl, C1-C6 alkoxy, or substituted C1-C6 alkoxy; R 6A For H, N(R) a (R) b ), acetyl, formyl, or O-phenyl; Y 7A For N and R 7A It either does not exist or is a C1-C6 alkyl group; or Y 7A For C and R 7A Selected from H, C1-C6 alkyl, or CN(R) a (R) b );Y 8A For N and R 8A Does not exist, or Y 8A For C and R 8A Selected from H, halogens, OH, C1-C6 alkyl groups, or substituted C1-C6 alkyl groups; R a and Rb Independently selected from H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl, substituted C1-C6 alkenyl, acetyl, formyl, or together forming a 5-7 membered heterocycle; excluding Y. 7A For N, Y 8A For C, R 8A For H, R 2A Let H be the number of 'R', and R be the number of 'R'. 6A This refers to the case of NH2 (unmodified adenine).

[0045] In some implementations, the modified guanine has structure (II):

[0046] Among them, R 2G For N(R) a (R) b ); R 6G It is an oxygen-substituted (=O) and R 1G For H, or R 6G Selected from O-C1-C6 alkyl or S-C1-C6 alkyl and R 1G Does not exist; Y 7G For N and R 7A It is absent or is a C1-C6 alkyl group; or Y 7G For C and R 7G Selected from H, C1-C6 alkyl, or CN(R) a (R) b );Y 8G For N and R 8G Does not exist, or Y 8G For C and R 8G Selected from H, halogens, OH, C1-C6 alkyl groups, or substituted C1-C6 alkyl groups; R a and R b Independently selected from H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl, substituted C1-C6 alkenyl, acetyl, formyl, or together forming a 5-7 membered heterocycle; excluding Y. 7G For N, Y 8G For C, R 8G For H, R 2G It is NH2 and R 6G This is the case where it equals O (unmodified guanine).

[0047] In some embodiments, the modified thymine or modified uracil has structure (III):

[0048] Where X is selected from O or S, and R 5U Selected from H, OH, halogens, O-Cl-C 12 Alkyl, O-C1-C12 Substituted alkyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C1-C 12 Alkenyl, substituted C1-C 12 Alkenyl; where if each X is O, then R 5U It is neither H nor CH3 (unmodified uracil and unmodified thymine, respectively).

[0049] In some implementations, those skilled in the art can, depending on whether the nucleotide is RNA or DNA, add R... 5U H is replaced with CH3 or CH3 is replaced with H (that is, the thymine and uracil are interchanged).

[0050] In some implementations, the modified cytosine has structure (IV):

[0051] Where X is selected from O or S, R 4C For N(R) a (R) b ); R 5C Selected from H, OH, halogens, O-Cl-C 12 Alkyl, O-C1-C 12 Substituted alkyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C1-C 12 Alkenyl, substituted C1-C 12 alkenyl; R a and R b Independently selected from H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl, substituted C1-C6 alkenyl, acetyl, formyl, or together forming a 5-7 membered heterocycle; excluding X being O, R 4C It is NH2 and R 5C The case is H (unmodified cytosine).

[0052] Preferably, the base modification may include methylation modification at any site of purine or pyrimidine in any nucleotide. Specifically, for example, introducing a methyl group (-CH3) at the 5' position (the 5th carbon atom) of a pyrimidine, the base with this base modification is called 5-methylcytosine, denoted as (5m)C in the sequence;

[0053] Alternatively, the base modification can also be achieved by modifying to form any of the following modified bases: 2-thiouracil (“2-thioU”), 2-thiocytosine (“2-thioC”), 4-thiouracil (“4-thioU”), 6-thioguanine (“6-thioG”), 2-aminoadenine (“2-aminoA”), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazoguanine, 7-deazo-8-azaguanine, 7-deazoadenine, 7-deazo-8-azaadenine, 5-methyluracil (“5-methylU”), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyl Uracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil (“5-allyl U”), 5-allylcytosine (“5-allyl C”), 5-aminoallyluracil (“5-aminoallyl U”), 5-aminoallyl-cytosine (“5-aminoallyl C”), debased nucleotides, Z bases, P bases, non-structural nucleic acids (“UNA”), isoguanine (“isoG”), isocytosine (“isoC”), glycerol nucleic acid (GNA), glycerol nucleic acid (GNA), thiomorpholine (C4H9NS) or thiophosphoramide morpholine (TMO), pseudoisocytosine, 5-bromouracil, inosine, and 2-chloro-6-aminopurine.

[0054] Preferably, the modification of the phosphate backbone can be thiophosphate modification, methylated phosphate modification, dimercaptophosphate modification, or 5'-(E)-VP modification. Preferably, the modification of the phosphate backbone can also be achieved by forming other types of phosphate ester bonds, including but not limited to methyl phosphate, selenophosphate, methylboryl phosphate, and dithiophosphate.

[0055] In some embodiments, the ribose modification may be a non-bicyclic modified glycosyl moiety. In some embodiments, the modified glycosyl moiety is a bicyclic or tricyclic glycosyl moiety. In some embodiments, the sugar-modified nucleotide contains a cyclopropyl group at the 5' carbon of the sugar. In some embodiments, the sugar-modified nucleotide contains a bridge between the 4'-carbon and 2'-carbon of the sugar, and the sugar-modified nucleotide is a 4'-2'-bridged bicyclic nucleotide. In some embodiments, the modified glycosyl moiety is a non-bicyclic modified furanyl glycosyl moiety containing one or more acyclic substituents, including but not limited to substituents at the 2', 3', 4', and / or 5' positions. In some embodiments, the furanyl glycosyl moiety is a ribosyl glycosyl moiety. In some embodiments, one or more acyclic substituents of the non-bicyclic modified glycosyl moiety are branched. In some embodiments, the modified glycosyl moiety contains one or more unbridged sugar substituents and one or more bridging sugar substituents (e.g., a 5'-substituted and 4'-2'-bridged sugar).

[0056] In some embodiments, the ribose modification includes ribose 2' substitution modification, wherein the 2' substituent is independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, or halogen.

[0057] In some embodiments, the 2'-substituent is selected from: halogenated, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, O-C1-C. 10 Alkoxy, O-C1-C10 substituted alkoxy, O-C1-C 10 Alkyl, O-C1-C 10 Substituted alkyl, S-alkyl, N(R) m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-Alkenyl, O-alkynyl, S-alkynyl, N(R m )-Alkyne, O-alkylene-O-alkyl, Alkyne, Alkylaryl, Arylalkyl, O-Alkylaryl, O-Arylalkyl, O(CH2)2SCH3, O(CH2)2ON(R m (R) n ) or OCH2C(=O)-N(R m (R) n ), where each R m and R n Independently H, amino protecting group, or substituted or unsubstituted C1-C 10 Alkyl, -O(CH2)2ON(CH3)2 ("DMAOE"), or 2'-O(CH2)2O(CH2)2N(CH3)2 ("DMAEOE"). Some embodiments of the above 2'-substituents may be further substituted by one or more substituents independently selected from the following: hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro(NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0058] Preferably, the ribose modification includes ribose 2' substitution modification, specifically, the 2' position of the ribose has any one of the following substituents: allyl, amino, azide, thio, O-allyl, O-Cl-C 10 Alkyl, -OCF3, O-(CH2)2-O-CH3, -O(CH2)2SCH3, O-(CH2)2-ON(R) m (R) n ), or O-CH2-C(=O)-N(R m (R) n ), where each R m and R n Independently H or substituted or unsubstituted C1-C 10 alkyl.

[0059] In some embodiments, the ribose modification may be a sugar substitute. Such a sugar substitute may contain one or more substituents corresponding to substituents in other types of modifications to the glycosyl moiety.

[0060] In some embodiments, the oxygen atom of the glycosyl moiety is substituted, for example, with a sulfur, carbon, or nitrogen atom. In such embodiments, the modified glycosyl moiety also contains bridging and / or unbridging substituents. For example, some sugar substitutes contain a 4'-sulfur atom and substitutions at the 2'- and / or 5'-positions.

[0061] In some embodiments, the sugar substitute comprises a ring having a number of atoms other than five. For example, in some embodiments, the sugar substitute comprises a six-membered tetrahydropyran (THP) or a modified or substituted THP.

[0062] In some embodiments, the modified THP nucleoside is represented by the following general formula:

[0063] Wherein, Bx is the nucleobase moiety; T3 and T4 are each independently an internucleotide linker group that connects the modified THP nucleoside to the rest of the oligonucleotide, or one of T3 and T4 is an internucleotide linker group that connects the modified THP nucleoside to the rest of the oligonucleotide, and the other T3 or T4 is H, a hydroxyl protecting group, a linked conjugation group, or a 5' or 3'-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted The C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and R1 and R2 are each independently selected from: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2 and J3 is independently H or C1-C6 alkyl.

[0064] In some embodiments, q1, q2, q3, q4, q5, q6, and q7 of the modified THP nucleoside are each H. In some embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is not H. In some embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In some embodiments, one of R1 and R2 of the modified THP nucleoside is F. In some embodiments, R1 is F and R2 is H; in some embodiments, R1 is methoxy and R2 is H; in some embodiments, R1 is methoxyethoxy and R2 is H.

[0065] In some embodiments, the sugar substitute comprises a ring having more than five atoms and more than one heteroatom. For example, a nucleoside comprising a morpholine glycosyl moiety. The term "morpholine" refers to a sugar substitute having the following structure:

[0066] Wherein, Bx is the nucleobase moiety; the morpholine structure can be further modified, for example, by adding or changing various substituents to the above morpholine structure.

[0067] In some embodiments, the sugar substitute comprises an acyclic moiety. Examples of nucleosides and oligonucleotides comprising such acyclic sugar substitutes include, but are not limited to, peptide nucleic acids (PNAs), acyclic butyl nucleic acids, and other nucleosides and oligonucleotides described herein.

[0068] In some implementations, the sugar substitute is an unlocked nucleic acid nucleotide (UNA) or a glycol nucleic acid nucleotide (GNA).

[0069] In some embodiments, the phosphate backbone modification includes phosphate backbone modification of the spacer segment and phosphate backbone modification of the wing segment.

[0070] In some embodiments, the nucleosides of the antisense oligonucleotide can be linked using one or more modified internucleotide linkages. Representative phosphorus-containing internucleotide linkages include, but are not limited to, phosphate diesters (containing phosphate diester bonds), phosphate triesters, methylphosphonates, phosphoramide esters, thiophosphates, and dithiophosphates. Representative non-phosphorus-containing internucleotide linkage groups include, but are not limited to, methylmethylimino (-CH2-N(CH3)-O-CH2-), thiodies, thiocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to natural phosphate diester internucleotide linkages, modified internucleotide linkages can be used to alter (generally increase) the nuclease resistance of the oligonucleotide. This disclosure uses different modified internucleotide linkages, such as thiophosphate and / or methanesulfonylphosphonamide linkages, all of which achieve similar targeting effects on renal tubules.

[0071] In some embodiments, the internucleotide linker with chiral atoms can be prepared as a racemic mixture or as individual enantiomers. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleotide links are well known to those skilled in the art.

[0072] In some implementations, the modified nucleoside linker has the general formula Z1, Z2, or Z3:

[0073] Where each X 1 Independently selected from O and S; X 2 Selected from O, NR1, CH2 and S; X 3 Selected from O, NR 1 CH2 and S; L does not exist, or NR 1 、N(R 1 SO2, –N=, O, C1-C6 alkylene, or C1-C6 heteroalkylene; each R 1 Independently selected from H, C1-C6 alkyl, and substituted C1-C6 alkyl, or two R on the same atom 1 Together they form = O; and R 2 Selected from -OH, -SH, Cl-C 22 Alkyl, substituted C1-C 22 Alkyl, C2-C 22 Alkenyl, substituted C2-C 22 Alkenyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, aryl, and substituted aryl; wherein when the group is substituted, it contains one or more substituents selected from: halogen, -OH, N(R) 1 )2、-O-C1-C6 alkyl、C1-C 22 Alkyl, C2-C 22 Alkenyl, cycloalkyl, heterocyclic, heteroaryl, and aryl.

[0074] In some implementations, the modified nucleoside linker comprises the general formula Z4:

[0075] Wherein, X is selected from O or S; R1 is selected from H, C1-C6 alkyl, and substituted C1-C6 alkyl; and T is selected from SO2R2, C(=O)R3, and P(=O)R4R5, wherein: R2 is selected from aryl, substituted aryl, heterocyclic, substituted heterocyclic, aromatic heterocyclic, substituted aromatic heterocyclic, diazole, substituted diazole, C1-C6 alkoxy, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, substituted C1-C6 alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkynyl, and conjugated groups; R3 is selected from aryl, substituted aryl, CH3, N(CH3)2, OCH3 and conjugated groups; R4 is selected from OCH3, OH, C1-C6 alkyl, substituted C1-C6 alkyl and conjugated groups; and R5 is selected from OCH3, OH, C1-C6 alkyl, and substituted C1-C6 alkyl.

[0076] In some embodiments, the modified internucleotide linkage comprises a mesylate phosphoramidate internucleoside linkage (MsPA) group, which has the general formula:

[0077] The thiophosphate and / or methanesulfonylphosphatidyl linkage contains a chiral center.

[0078] In some embodiments, the antisense oligonucleotide comprises the general formula (R p ) and / or (S p The methanesulfonyl phosphoramide ester shown is represented by the symbol "B", where "B" indicates a nucleobase.

[0079] In some embodiments, the antisense oligonucleotide comprises the general formula (R p ) and / or (S p The thiophosphate ester shown is an example where "B" represents a nucleobase:

[0080] In some embodiments, representative internucleotide linkages with chiral centers include, but are not limited to, alkylphosphonates and thiophosphates. Modified oligonucleotides containing internucleotide linkages with chiral centers can be prepared as antisense oligonucleotides containing stereorandom internucleotide linkages, or as antisense oligonucleotides containing such internucleotide linkages having a specific stereochemical configuration.

[0081] In some embodiments, the internucleotide linker with chiral atoms can be prepared as a racemic mixture or as individual enantiomers. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleotide links are well known to those skilled in the art.

[0082] In some embodiments, the antisense oligonucleotide comprises a phosphate thioester nucleoside linker, wherein all phosphate thioester nucleoside linkers are stereo-random. In some embodiments, the antisense oligonucleotide comprises a methanesulfonylphosphatidyl ester nucleoside linker, wherein all methanesulfonylphosphatidyl ester nucleoside linkers are stereo-random. Such modified oligonucleotides can be produced using synthetic methods that result in random selection of the stereoconfiguration of each chiral linker. Nevertheless, each chiral linker of each individual oligonucleotide molecule has a defined stereoconfiguration.

[0083] In some embodiments, the antisense oligonucleotide is enriched with a modified oligonucleotide comprising one or more specific thiophosphate and / or methanesulfonylphosphatidyl ester nucleoside linkages, each linkage being independently in a specific, independently selected stereochemical configuration. In some embodiments, the specific configuration of the specific thiophosphate and / or methanesulfonylphosphatidyl ester linkage is present in at least 65% of the molecules in the population. In some embodiments, the specific configuration is present in at least 70% of the molecules in the population. In some embodiments, the specific configuration is present in at least 80% of the molecules in the population. In some embodiments, the specific configuration is present in at least 90% of the molecules in the population. In some embodiments, the specific configuration is present in at least 99% of the molecules in the population. Such chiral-enriched antisense oligonucleotides can be produced using synthetic methods known in the art.

[0084] In some embodiments, the antisense oligonucleotide is enriched with at least one designated thiophosphate ester and / or methanesulfonylphosphatidyl ester in (S p Modified oligonucleotides with a (R) configuration. In some embodiments, the antisense oligonucleotide is enriched with at least one thiophosphate ester and / or methanesulfonylphosphatidyl ester in the (R) configuration. p Oligonucleotides with modified configurations.

[0085] Unless otherwise stated, the internucleotide linkages with chiral centers in the antisense oligonucleotides described herein may be stereo-random or in a specific stereochemical configuration.

[0086] In some embodiments, the antisense oligonucleotide comprises a neutral nucleoside linker, including but not limited to, phosphate triesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), methyl acetal (3'-O-CH2-O-5'), methoxypropyl (MOP), and thiomethyl acetal (3'-S-CH2-O-5'). Further neutral nucleoside linkers include nonionic links comprising siloxanes (dialkylsiloxanes), carboxylic esters, carboxamides, sulfides, sulfonates, and amides. Further neutral nucleoside linkers include nonionic links comprising a mixture of N, O, S, and CH2 components.

[0087] In some embodiments, the antisense oligonucleotide comprises one or more reverse nucleosides, as shown below:

[0088] Each Bx represents any nucleobase independently.

[0089] In some implementations, nucleotides can be linked by 2'-5' bonds, as shown below:

[0090] Each Bx represents any nucleobase independently.

[0091] In one specific embodiment, the internucleotide bonds of the antisense oligonucleotides are modified with thiophosphate (also known as thiomodification), and the nucleotides are linked by thiophosphate ester bonds.

[0092] In one specific embodiment, the internucleotide bonds of the antisense oligonucleotide include thiophosphate bonds and / or methanesulfonylphosphatidyl linkages.

[0093] In one specific embodiment, the internucleotide bond of the antisense oligonucleotide simultaneously includes at least one thiophosphate bond and at least one methanesulfonylphosphatidyl linkage.

[0094] In one specific embodiment, the nucleotides in the spacer region have deoxyribose, and the deoxyribose has a hydrogen (-H) at the 2' position.

[0095] In one specific embodiment, the bases of the nucleotides in the antisense oligonucleotide are selected from any one or more of the following: adenine (A), thymine (T), guanine (G), and 5-methylcytosine ((5m)C).

[0096] Preferably, the antisense oligonucleotide targets a target gene.

[0097] Preferably, the antisense oligonucleotide is at least partially complementary to any segment of nucleotides in the transcribed mRNA of the target gene; specifically, the mismatch between the antisense oligonucleotide and any segment of the target gene does not exceed 5, 4, 3, 2, or 1 nucleotide; preferably, the antisense oligonucleotide is completely complementary to any segment of nucleotides in the transcribed mRNA of the target gene.

[0098] Preferably, the target gene is a gene expressed in proximal renal tubular cells; preferably, the target gene may include one or more of SGLT2, URAT1, and MALAT1.

[0099] Preferably, the base sequence of the antisense oligonucleotide comprises a base sequence that differs from the base sequence of the nucleotide shown in any one of SEQ ID NO. 15, 16, 19, 20, 24, 25, 26, 28 by no more than 4, 3, 2, or 1 bases.

[0100] Preferably, the base sequence of the antisense oligonucleotide comprises the base sequence of the nucleotide shown in any one of SEQ ID NO. 15, 16, 19, 20, 24, 25, 26, 28, or the base sequence of the antisense oligonucleotide is as shown in any one of SEQ ID NO. 15, 16, 19, 20, 24, 25, 26, 28.

[0101] Preferably, the antisense oligonucleotide is selected from any one of the following:

[0102] (1) Having the base sequence of the nucleotides shown in SEQ ID NO.15 and the modification pattern shown in 2-8-2;

[0103] (2) Having the base sequence of the nucleotides shown in SEQ ID NO.16 and the modification pattern shown in 2-8-2;

[0104] (3) Having the base sequence of the nucleotides shown in SEQ ID NO.19 and the modification pattern shown in 2-8-3;

[0105] (4) Having the base sequence of the nucleotides shown in SEQ ID NO.20 and the modification pattern shown in 2-8-2;

[0106] (5) Having the base sequence of the nucleotides shown in SEQ ID NO.24 and the modification pattern shown in 2-8-3;

[0107] (6) Having the base sequence of the nucleotides shown in SEQ ID NO.25 and the modification pattern shown in 2-8-2;

[0108] (7) Having the base sequence of the nucleotides shown in SEQ ID NO.26 and the modification pattern shown in 2-8-2; or

[0109] (8) Having the base sequence of the nucleotides shown in SEQ ID NO.28 and the modification pattern shown in 2-8-2;

[0110] In the modified pattern, the first number represents the number of nucleotides in the 5' wing region, the second number represents the number of nucleotides in the spacer region, and the third number represents the number of nucleotides in the 3' wing region.

[0111] In one specific embodiment, the structure of the antisense oligonucleotide is a 5' wing region-spacer region-3' wing region, wherein the 5' wing region consists of two nucleotides with a first chemical modification, the 3' wing region consists of two nucleotides with a second chemical modification, and the spacer region consists of eight modified or unmodified nucleotides; the first chemical modification is the connection of -CH2-O- between the 4' and 2' positions of the ribose, and the second chemical modification is the connection of -CH2-O- between the 4' and 2' positions of the ribose; the nucleotides in the spacer region have deoxyribose, and the bases of the nucleotides in the antisense oligonucleotide include any one or more of the following: A, T, (5m)C, and G; the nucleotides in the antisense oligonucleotide are linked by phosphate thioester bonds.

[0112] More specifically, the antisense oligonucleotide includes any of the following:

[0113] (1)LNA-G* / LNA-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / LNA-T* / LNA-(5m)C(SEQ ID NO.1)(ASO-LNA)

[0114] (2)LNA-A* / LNA-G* / dA* / dT* / d(5m)C* / dT* / dT* / dG* / dG* / dT* / LNA-G* / LNA-A(SEQ ID NO.6)(ASO_13)

[0115] (3)LNA-A* / LNA-G* / dA* / dA* / dT* / dG* / d(5m)C* / dG* / dG* / d(5m)C* / LNA-T* / LNA-G(SEQ ID NO.11)(ASO_96)

[0116] (4)LNA-(5m)C* / LNA-(5m)C* / dA* / dG* / dG* / dA* / dG* / dT* / dT* / d(5m)C* / LNA-A* / LNA-G(SEQ ID NO.12)(ASO_348_12)

[0117] (5)LNA-A* / LNA-G* / dT* / dT* / d(5m)C* / dA* / d(5m)C* / dT* / dG* / dA* / LNA-A* / LNA-T(SEQ ID NO.14)(ASO-Malat1)

[0118] In this context, LNA- represents the connection between the 4' and 2' positions of the ribose, -CH2-O-, * represents the connection between nucleotides via a thiophosphate bond, (5m) indicates that the 5' carbon in the cytosine base is methylated, d represents that the ribose is deoxyribose, and each nucleotide is separated by " / ".

[0119] In one specific embodiment, the structure of the antisense oligonucleotide is a 5' wing region-spacer region-3' wing region, wherein the 5' wing region consists of two nucleotides with a first chemical modification, the 3' wing region consists of two nucleotides with a second chemical modification, and the spacer region consists of eight modified or unmodified nucleotides; the first chemical modification is the connection of -CH(CH3)-O- between the 4' and 2' positions of the ribose, and the second chemical modification is the connection of -CH(CH3)-O- between the 4' and 2' positions of the ribose; the nucleotides in the spacer region have deoxyribose, and the bases of the nucleotides in the antisense oligonucleotide include any one or more of the following: A, T, (5m)C, and G; the nucleotides in the antisense oligonucleotide are linked by phosphate thioester bonds.

[0120] More specifically, the antisense oligonucleotide is:

[0121] cET-G* / cET-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / cET-T* / cET-(5m)C(SEQ ID NO.2)(ASO-cEt)

[0122] Wherein, cET- represents the connection between the 4' and 2' positions of the ribose -CH(CH3)-O-, * represents the connection between nucleotides via a thiophosphate bond, (5m) indicates that the 5' carbon in the cytosine base is methylated, d represents that the ribose is deoxyribose, and each nucleotide is separated by " / ".

[0123] In one specific embodiment, the structure of the antisense oligonucleotide is a 5' wing region-spacer region-3' wing region, wherein the 5' wing region consists of two nucleotides with a first chemical modification, the 3' wing region consists of three nucleotides with a second chemical modification, and the spacer region consists of eight modified or unmodified nucleotides; the first chemical modification is the connection of -CH2-O- between the 4' and 2' positions of the ribose, and the second chemical modification is the connection of -CH2-O- between the 4' and 2' positions of the ribose; the nucleotides in the spacer region have deoxyribose, and the bases of the nucleotides in the antisense oligonucleotide include any one or more of the following: A, T, (5m)C, and G; the nucleotides in the antisense oligonucleotide are linked by phosphate thioester bonds.

[0124] More specifically, the antisense oligonucleotide includes any of the following:

[0125] (1)LNA-A* / LNA-G* / dA* / dT* / d(5m)C* / dT* / dT* / dG* / dG* / dT* / LNA-G* / LNA-A* / LNA-A(SEQ ID NO.5)(ASO_12)

[0126] (2)LNA-A* / LNA-G* / dA* / dA* / dT* / dG* / d(5m)C* / dG* / dG* / d(5m)C* / LNA-T* / LNA-G* / LNA-A(SEQ ID NO.10)(ASO_95)

[0127] In this context, LNA- represents the connection between the 4' and 2' positions of the ribose, -CH2-O-, * represents the connection between nucleotides via a thiophosphate bond, (5m) indicates that the 5' carbon in the cytosine base is methylated, d represents that the ribose is deoxyribose, and each nucleotide is separated by " / ".

[0128] Preferably, the antisense oligonucleotide is 12 nucleotides in length.

[0129] Preferably, the antisense oligonucleotide is selected from any one of the following:

[0130] (1) Having the base sequence of the nucleotides shown in SEQ ID NO.15 and the modification pattern shown in 2-8-2;

[0131] (2) Having the base sequence of the nucleotides shown in SEQ ID NO.16 and the modification pattern shown in 2-8-2;

[0132] (3) Having the base sequence of the nucleotides shown in SEQ ID NO.20 and the modification pattern shown in 2-8-2;

[0133] (4) Having the base sequence of the nucleotides shown in SEQ ID NO.25 and the modification pattern shown in 2-8-2;

[0134] (5) Having the base sequence of the nucleotides shown in SEQ ID NO.26 and the modification pattern shown in 2-8-2; or

[0135] (6) Having the base sequence of the nucleotides shown in SEQ ID NO.28 and the modification pattern shown in 2-8-2;

[0136] In the modified pattern, the first number represents the number of nucleotides in the 5' wing region, the second number represents the number of nucleotides in the spacer region, and the third number represents the number of nucleotides in the 3' wing region.

[0137] Preferably, the antisense oligonucleotide is selected from any one of the following:

[0138] (1)LNA-G* / LNA-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / LNA-T* / LNA-(5m)C(SEQ ID NO.1),

[0139] (2)cET-G* / cET-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / cET-T* / cET-(5m)C(SEQ ID NO.2),

[0140] (3)LNA-A* / LNA-G* / dA* / dT* / d(5m)C* / dT* / dT* / dG* / dG* / dT* / LNA-G* / LNA-A (SEQ ID NO. 6),

[0141] (4)LNA-A* / LNA-G* / dA* / dA* / dT* / dG* / d(5m)C* / dG* / dG* / d(5m)C* / LNA-T* / LNA-G(SEQ ID NO.11),

[0142] (5)LNA-(5m)C* / LNA-(5m)C* / dA* / dG* / dG* / dA* / dG* / dT* / dT* / d(5m)C* / LNA-A* / LNA-G(SEQ ID NO.12), or

[0143] (6)LNA-A* / LNA-G* / dT* / dT* / d(5m)C* / dA* / d(5m)C* / dT* / dG* / dA* / LNA-A* / LNA-T (SEQ ID NO. 14),

[0144] In this context, LNA- represents the connection between the 4' and 2' positions of the ribose, -CH2-O-; cET- represents the connection between the 4' and 2' positions of the ribose, -CH(CH3)-O-; * represents the connection between nucleotides via a thiophosphate bond; (5m) indicates that the 5' carbon in the cytosine base is methylated; d represents that the ribose is deoxyribose; and each nucleotide is separated by a " / ".

[0145] On the other hand, this disclosure also provides an antisense oligonucleotide conjugate comprising the aforementioned antisense oligonucleotide or a pharmaceutically acceptable salt thereof and a targeting ligand.

[0146] Preferably, the targeting ligand is conjugated to the antisense oligonucleotide.

[0147] Preferably, the targeting ligand is conjugated to the 3' or 5' end of the antisense oligonucleotide.

[0148] Preferably, the targeting ligand includes galactosamine, polypeptide, protein, sterol, lipid, phospholipid, biotin, phenoxazine, active drug, cholesterol, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, folic acid, dye, and sugar.

[0149] Preferably, the targeting ligand can be a protein, which includes an antibody or antigen-binding fragment. Specifically, the antigen-binding fragment may include IgG, Fab, scFv, single-domain antibodies, immunoglobulin fragments, one or more VHH domains, etc. Antisense oligonucleotides are conjugated to the antibody or antigen-binding fragment to form antibody-oligonucleotide conjugates.

[0150] Preferably, the conjugated portion can be a lipid. Examples of lipids include, but are not limited to, cholesterol moieties, thioethers (e.g., hexyl-S-triphenylmethylthiol), thiocholesterol, aliphatic chains (e.g., dodecyl glycol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or 1-di-O-hexadecyl-rac-tripropyl-SH-triethylammonium phosphonate), polyamines or polyethylene glycol chains, adamantaneacetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-hydroxycholesterol moieties.

[0151] Preferably, the conjugated portion may be an active pharmaceutical ingredient. Examples of such active pharmaceutical ingredients include, but are not limited to, aspirin, warfarin, phenylbutazone, ibuprofen, sulprofen, fenbufen, ketoprofen, (5)-(+)-pranoprofen, carboprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, leucovorin, benzothiazide, chlorothiazide, diazepam, indomethacin, barbiturates, cephalosporins, sulfonamides, antidiabetic drugs, antibacterial drugs, or antibiotics.

[0152] Preferably, the conjugated portion can be a sugar, especially an amino sugar, more specifically N-acetylgalactosamine (GalNAc).

[0153] Preferably, the conjugated portion may be a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a higher valent GalNAc portion.

[0154] Preferably, the joining portion can be attached to the ASO via 1, 2, 3, 4 or 5 or more connectors.

[0155] Preferably, the connector includes a phosphate diester (p or po) connector, a thiophosphate (ps) connector, a methanesulfonyl phosphate amide (yp) connector, a phosphorous amide (HEG) connector, a triethylene glycol (TEG) connector, a dithiophosphate connector, p-(PS)2, (PS)2-p-TEG-p, (PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

[0156] Preferably, the targeting ligand comprises a protein, a small molecule compound, a carbohydrate, or a lipid.

[0157] Preferably, the protein includes natural proteins, synthetic polyamino acids, polypeptides, and antibodies.

[0158] Preferably, the natural protein includes human serum albumin, low-density lipoprotein, or globulin.

[0159] Preferably, the synthesized polyamino acids include polylysine, poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactic acid-co-ethylene glycol) copolymer, diethylene ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer, polyethylene glycol, polyvinyl alcohol, polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphoric acid.

[0160] Preferably, the carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, amino sugars, or hyaluronic acid.

[0161] Preferably, the lipids include fatty acids, sterols, or phospholipids;

[0162] Preferably, the fatty acids include capric acid, caprylic acid, lauric acid, palmitic acid, myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, or eicosenoic acid.

[0163] Preferably, the sterol includes cholesterol, cholesterol group, cholesterol alcohol, stigmasterol, cholesterol acid, or ergosterol.

[0164] Preferably, the phospholipid comprises di-hexadecyl-racemic glycerol or triethyl-amine 1,2-di-O-hexadecyl-racemic glycerol-3-hydrophosphonate.

[0165] Preferably, the targeting ligand comprises amino sugars.

[0166] Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0167] Preferably, the GalNac portion can be a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion.

[0168] Preferably, the targeting ligand has the following structure:

[0169] On the other hand, this disclosure also provides a composition containing the aforementioned antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the composition containing the aforementioned antisense oligonucleotide conjugate.

[0170] Preferably, the composition further contains a pharmaceutically acceptable carrier.

[0171] On the other hand, this disclosure also provides a cell containing the aforementioned antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned antisense oligonucleotide conjugate, or the aforementioned composition.

[0172] On the other hand, this disclosure also provides any one of the following applications of the aforementioned antisense oligonucleotide or its pharmaceutically acceptable salt, the aforementioned antisense oligonucleotide conjugate, the aforementioned composition, or the aforementioned cells:

[0173] (1) Use in the preparation of medicines for the prevention and / or treatment of diseases.

[0174] (2) Application in suppressing target gene expression

[0175] (3) Application in targeting proximal renal tubules.

[0176] On the other hand, this disclosure also provides the use of the aforementioned antisense oligonucleotides or their pharmaceutically acceptable salts, the aforementioned antisense oligonucleotide conjugates, the aforementioned compositions, or the aforementioned cells in the prevention and / or treatment of diseases.

[0177] On the other hand, the present invention provides a method for preventing and / or treating a disease, the method comprising administering to a subject an effective amount of the aforementioned antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the aforementioned antisense oligonucleotide conjugate, the aforementioned composition or the aforementioned cells.

[0178] On the other hand, this disclosure also provides a method for reducing target gene expression, the method comprising contacting the aforementioned antisense oligonucleotide, the aforementioned antisense oligonucleotide conjugate, the aforementioned composition, or the aforementioned cells with the target gene. Preferably, the inhibition of target gene expression refers to the inhibition of target gene expression in proximal renal tubular cells. Preferably, the method is not for therapeutic purposes. Preferably, the method is performed in vitro.

[0179] On the other hand, this disclosure provides the use of the aforementioned antisense oligonucleotides, the aforementioned antisense oligonucleotide conjugates, the aforementioned compositions or the aforementioned cells for inhibiting target genes in targeting proximal renal tubules, preventing and / or treating diseases and / or inhibiting target gene expression.

[0180] Preferably, the disease includes diseases caused by abnormal expression of any one or more of the following genes: SGLT2, URAT1, or MALAT1.

[0181] Preferably, the target gene is a gene expressed in proximal renal tubular cells; specifically, the target gene may include one or more of SGLT2, URAT1, or MALAT1.

[0182] Preferably, the proximal renal tubule includes a proximal convoluted tubule and / or a proximal straight tubule.

[0183] Preferably, the proximal renal tubular cells include proximal convoluted tubular cells and / or proximal straight tubular cells. Detailed Implementation

[0184] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided through specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of this disclosure. All other implementations obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0185] This disclosure may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this disclosure may be combined with technical features of any or more other embodiments to obtain further embodiments. This disclosure includes such further embodiments obtained through combination.

[0186] (I) Definitions and Explanations

[0187] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used have meanings commonly understood by those skilled in the art. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this disclosure are all widely used terms and routine procedures in their respective fields. It should also be understood that the terminology used in this disclosure is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0188] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and that used in this disclosure, the use and terminology of this disclosure shall prevail. Section headings used in this disclosure are for organizational purposes only and should not be construed as limiting the subject matter.

[0189] In this disclosure, the conjunction term “and / or” between multiple elements means to include both the meaning of “and” and “or”, for example, the phrase “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0190] In this disclosure, the terms “comprising,” “including,” “having,” and “containing,” and any variations thereof, are intended to cover non-exclusive inclusion. The term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially composed of.” In this disclosure, the term “containing” indicates that various ingredients may be used together in mixtures or compositions of this disclosure. Therefore, the terms “substantially composed of” and “composed of” are included in the term “containing.”

[0191] The range of numbers used in this disclosure should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0192] In this disclosure, the terms “about” or “approximately” applied to one or more target values ​​refer to values ​​similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the terms “approximately” or “about” refer to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0193] In this disclosure, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0194] In this disclosure, the disclosure of all ranges should be considered as a disclosure of all subranges and all point values ​​within the range. All point values ​​can be combined independently. Since these ranges are continuous, they include every numerical value between the minimum and maximum values. It should also be understood that any numerical range referenced in this application is intended to include all subranges within that range. For example, the disclosure of 0.1-200 should be considered as also disclosing ranges such as 0.1-10, 1-100, 100-200, etc., and also disclosing point values ​​such as 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200.

[0195] In this disclosure, references to "some implementations" or "some implementation methods" describe a subset of all possible embodiments.

[0196] In this disclosure, the term "base" or "nucleobase" refers to the basic building blocks of nucleosides, nucleotides, and nucleic acids, containing nitrogen as a constituent element; it is also called a "nitrogenous base." Nucleobases are mainly divided into two categories: purines and pyrimidines. In this disclosure, the term "purine" is formed by the fusion of a six-membered pyrimidine ring and a five-membered imidazole ring, forming a bicyclic structure (containing four nitrogen atoms located at positions 1, 3, 7, and 9). In nucleic acids, purine derivatives include adenine and guanine. In this disclosure, the term "pyrimidine" is a heterocyclic compound formed by two nitrogen atoms replacing two carbon atoms at the metaposition of a benzene molecule. In nucleic acids, pyrimidine derivatives include three nucleobases: cytosine, thymine, and uracil. Unless otherwise specified, the capital letters A, U, T, G and C in this article represent the base composition of nucleotides, namely adenine (A), uracil (U), thymine (T), guanine (G) and cytosine (C).

[0197] In this disclosure, the term "modified base" means any base other than adenine, cytosine, guanine, thymine, or uracil.

[0198] In this disclosure, the term "nucleoside" refers to a compound consisting of a base (purine or pyrimidine) and either ribose or deoxyribose.

[0199] In this disclosure, the term "nucleotide" refers to a compound consisting of a base (purine or pyrimidine), ribose or deoxyribose, and a phosphate group.

[0200] In this disclosure, the term "modified nucleotide" means a nucleotide that has a modified sugar moiety, a modified nucleoside internucleotide bond, and / or a modified base.

[0201] In this disclosure, the term "deoxyribose" refers to ribose with a hydrogen atom at the 2' position of its pentose sugar compared to ribose. In the sequences of this disclosure, d represents that the ribose is deoxyribose, such as dA, which indicates that the nucleotide has a deoxyribose and a base A (adenine), and if not specified, it indicates that the pentose sugar has a hydroxyl group (-OH) at the 2' position.

[0202] In this disclosure, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) having a length of, for example, less than 100, 200, 300, or 400 nucleotides. Oligonucleotides may comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides or modified deoxyribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region.

[0203] In this disclosure, the term "antisense oligonucleotide (ASO)" refers to an oligomer or polymer of nucleotides, such as naturally occurring nucleotides or their modified forms, which are covalently linked to each other by nucleotide-nucleotide bonds. The ASO is complementary to the target nucleic acid, enabling the ASO to hybridize with the target nucleic acid sequence.

[0204] In this disclosure, the term "complementary" refers to a structural relationship between nucleotides that allows them to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of a nucleic acid can be base-paired together by forming hydrogen bonds. In some embodiments, complementary nucleotides may be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable double helix. Those skilled in the art will recognize that the antisense oligonucleotides in this disclosure are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% complementary to the target nucleic acid. The term "complementary" in this disclosure may include up to about 20% mismatched nucleotides. Antisense oligonucleotides contain no more than about 20%, 15%, 10%, 5%, or no mismatched nucleotides. Those skilled in the art will recognize that it is possible to include mismatches while maintaining the binding capacity of the oligomeric compound.

[0205] In this disclosure, the term "mismatch" refers to a double-stranded nucleic acid where corresponding bases are not paired in a complementary manner. In cases where the antisense oligonucleotide is not perfectly complementary to the target gene sequence, the mismatch can be located within the antisense oligonucleotide or in its terminal regions. Typically, the most tolerant mismatches are located in the terminal regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0206] In this disclosure, the terms "identity" or "homology" have the same meaning and are used interchangeably, specifically referring to the sequence matching between two nucleic acids. When a position in two compared sequences is occupied by the same base (e.g., a position in each of two DNA molecules is occupied by adenine), then the molecules are identical at that position. The "percentage similarity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% similarity. For example, the DNA sequences CTGACT and CAGGTT have 50% similarity (three out of six positions match). Typically, two sequences are compared to produce the maximum similarity. Such alignments can be performed using methods known in the art. For example, the percentage similarity between two amino acid sequences can be determined using computer programs such as the Align program (DNAstar, Inc.), algorithms integrated into the ALIGN program, or by using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage similarity between two amino acid sequences can be determined using algorithms integrated into the GCG software package (available at www.gcg.com) with a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0207] In this disclosure, the term "target" refers to a protein that is intended to be regulated.

[0208] In this disclosure, the term "target gene" refers to the gene that encodes a target.

[0209] In this disclosure, the terms "target nucleic acid" and "nucleic acid molecule encoding a target" refer to any nucleic acid molecule whose expression and activity can be regulated by antisense oligonucleotides. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding a target (including, but not limited to, pre-mRNA and mRNA or portions thereof), cDNA derived from such RNA, and miRNA. For example, a target nucleic acid can be a cellular gene (or mRNA transcribed from such gene) whose expression is associated with a specific condition or disease state, or a nucleic acid molecule derived from an infectious agent.

[0210] In this disclosure, the terms “targeted” or “targeted to” refer to the binding of an antisense oligonucleotide to a specific target nucleic acid molecule or a specific region of a nucleotide within a target nucleic acid molecule.

[0211] Suitablely, the antisense oligonucleotides of the present invention can reduce the expression of target genes. In some embodiments, the antisense oligonucleotides of the present invention bind to the target nucleic acid and achieve at least 10% or 20% inhibition of expression compared to normal expression levels, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% inhibition compared to normal expression levels. The target gene expression level reaches at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

[0212] In this disclosure, the terms "modification" or "chemical modification" refer to chemical differences in a compound when compared to its naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar and base modifications) and phosphate backbone modifications. For the purposes of oligonucleotides, chemical modifications do not include differences solely in the base sequence.

[0213] In this disclosure, the terms "phosphate backbone modification" or "nucleoside inter-bond modification" refer to any alteration of naturally occurring nucleoside inter-bonds. "Naturally occurring nucleoside inter-bonds" refers to the 3' to 5' phosphodiester bond between nucleosides. The nucleoside inter-bond modifications include thiophosphate modification, methylphosphate modification, dimercaptophosphate modification, 5'-(E)-VP modification, methoxy modification, and fluorination modification. The nucleoside inter-bond modified by "thiophosphate (thio-modification)" is a thiophosphate ester nucleoside inter-bond, which refers to a bond between nucleosides with a non-bridging atom of sulfur, i.e., -OP(O)(S)O-.

[0214] In this disclosure, the term "base modification" can include methylation modification at any site of a purine or pyrimidine. Alternatively, modification can be performed to form any of the following modified bases: 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazoguanine, 7-deazo-8-azaguanine, 7-deazoadenine, 7-deazo-8-azaadenine, 5-methylcytosine ("5-methylC,(5m)C,5mC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynyl Cytosine, 5-propynyluracil, 5-ethynyluracil, 5-ethynyluracil, 5-allyluracil (“5-allyl U”), 5-allyluracil (“5-allyl C”), 5-aminoallyluracil (“5-aminoallyl U”), 5-aminoallyl-cytosine (“5-aminoallyl C”), debased nucleotides, Z bases, P bases, non-structural nucleic acids (“UNA”), isoguanine (“isoG”), isocytosine (“isoC”), glycerol nucleic acid (GNA), glycerol nucleic acid (GNA), thiomorpholine (C4H9NS) or thiophosphoramide morpholine (TMO), pseudoisocytosine, 5-bromouracil, inosine, and 2-chloro-6-aminopurine.

[0215] In this disclosure, the term "sugar modification" includes, but is not limited to: bridging two carbon atoms on a ribose (connecting one or more "linking groups" between the two carbon atoms) to form a sugar having a bicyclic structure, or substitution modification.

[0216] Specifically, the substitution modification occurs at the 2' position of the ribose and is referred to as "2' substitution modification". "2' substitution modification" refers to a sugar containing a non-H or non-OH substituent at the 2' position, and the substituent includes allyl, amino, azide, thio, O-allyl, and O-Cl-C. 10 Alkyl, -OCF3, O-(CH2)2-O-CH3, 2′-O(CH2)2SCH3, O-(CH2)2-ON(R) m (R) n ) or O-CH2-C(=O)-N(R m (R) n ), where R m and R n Independently H or substituted or unsubstituted C1-C 10 alkyl).

[0217] Specifically, the two carbon atoms bridging the ribose are connected by one or more "linking groups" between the 4' and 2' positions of the ribose. These "linking groups" include -[C(R1)(R2)]. n -, -C(R1)=C(R2)-, -C(R1)=N-, -C(=NR1)-, -C(=O)-, -C(=S)-, -O-, -Si(R1)2-, -S(=O) x -、-N(R1)-、-R-[C(R1)(R2)]n-R'-、-[C(R1)(R2)]nR-R'- or -R-R'-[C(R1)(R2)]n-、-RC(R1)=C(R2)-R'-、-R-R'-C(R1)=C(R2)-、-C(R1)=C(R2)-R-R'-。 Where R and R' independently include single bonds, -N(R3)-、-O-、-S-、-Se-、-Si(R4)(R5)-、-C(=O)-、-C(=S)-、-C(=NR3)-。

[0218] Preferably, the linking groups include -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -[C(R1)(R2)]nN(R3)-, [C(R1)(R2)]nN(R3)-O-, -[C(R1)(R2)]nON(R3)-, -[C(R1)(R2)]nS-, -C(R1)=C(R2)-, and -C(R1)=N-;

[0219] Where x is 0, 1, or 2;

[0220] n is 1, 2, 3 or 4;

[0221] R1, R2, R3, R4, and R5, each independently, include H, a protecting group, a hydroxyl group, and substituted or unsubstituted C1-C. 12 Alkyl, substituted or unsubstituted C 2- C 12 alkenyl, substituted or unsubstituted C2-C 12 Alkyne, substituted or unsubstituted C5-C 20 Aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroaryl, substituted or unsubstituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-J1), substituted acyl, CN, sulfonyl (S(=O)2-J1), sulfonyloxy (S(=O)-J1) or -C(=NH)-NH2, or R1 and R2 connected to form C3-C6 cycloalkyl or heterocyclic alkyl;

[0222] J1 and J2, independently, are H, substituted or unsubstituted C1-C.12 Alkyl, substituted or unsubstituted C2-C 12 alkenyl, substituted or unsubstituted C2-C 12 Alkyne, substituted or unsubstituted C5-C 20 Aryl, substituted or unsubstituted acyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted C1-C 12 Aminoalkyl or protecting group.

[0223] In this disclosure, the most preferred linking group is -CH2-O- or -CH(CH3)-O-.

[0224] In this disclosure, the term "locked nucleic acid (LNA)" or "LNA nucleotide" refers to a nucleic acid monomer that forms a bicyclic sugar bridge by a methylene (-CH2-) bridge between the 4' and 2' positions of the nucleotide sugar unit, and is selected from the following structures:

[0225] Where R is selected from O, S or N-R3, R1, R2 and R3 are defined as described above, and Base is a base.

[0226] In this disclosure, "Base" means base.

[0227] Specifically, in this disclosure, LNA refers to a bicyclic nucleotide analog, in which -CH2-O- is linked between the 4' and 2' positions of the ribose, and its structure is as follows:

[0228] Where Bx refers to a base.

[0229] In this disclosure, the nucleotide label for ribose-locked nucleic acids is LNA-Bx, where Bx can be any base; particularly, the bases A, G, C, T, and (5m)C. For example, LNA-G indicates that in this nucleotide, -CH2-O- is linked between the 4' and 2' positions of the ribose and the base is G (guanine).

[0230] In this disclosure, the term "restricted ethyl (cEt) modified nucleotide" refers to a bicyclic nucleotide analog with a -CH(CH3)-O- bond between the 4' and 2' positions of its ribose, and the structure of the restricted ethyl (cEt) modified nucleotide is as follows:

[0231] Where Bx refers to a base.

[0232] In this disclosure, nucleotides with ribose modified by a restrictive ethyl (cEt) group are labeled cET-Bx, where Bx can be any base; particularly, the bases A, G, C, T, and (5m)C. For example, cEt-G indicates that in this nucleotide, -CH(CH3)-O- is linked between the 4' and 2' positions of the ribose group, and the base is G (guanine).

[0233] In this disclosure, the term "(5m)C" refers to 5-methylcytosine, which is a product in which the 5' carbon atom of cytosine (C) is modified with a methyl group (-CH3).

[0234] In this disclosure, the term "hydroxyl group" alone or in combination refers to the -OH group.

[0235] In this disclosure, the term "carbonyl", alone or in combination, means a -C(O)- (-C(=O)-) group.

[0236] In this disclosure, the term "carboxy" alone or in combination refers to the -COOH group.

[0237] In this disclosure, the term "amino" alone or in combination means primary amino (-NH2), secondary amino (-NH-), or tertiary amino (-N-).

[0238] In this disclosure, the term "alkyl" refers, alone or in combination, to a straight-chain or branched alkyl group having 1 to 8 carbon atoms, particularly a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and even more particularly a straight-chain or branched alkyl group having 1 to 4 carbon atoms. Examples of straight-chain and branched C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl isomer, hexyl isomer, heptyl isomer, and octyl isomer, especially methyl, ethyl, propyl, butyl, and pentyl isomers. Specific examples of alkyl groups are methyl, ethyl, and propyl.

[0239] In this disclosure, the term "alkenyl," alone or in combination, refers to a straight-chain or branched hydrocarbon residue comprising an alkene bond and up to eight, preferably six, and particularly preferably four carbon atoms. Examples of alkenyl groups are vinyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, and isobutenyl.

[0240] In this disclosure, the term "alkynyl" alone or in combination means a straight-chain or branched hydrocarbon residue comprising a triple bond and up to eight, preferably six, and particularly preferably four carbon atoms.

[0241] In this disclosure, the term "aryl," alone or in combination, refers to a monovalent aromatic carbocyclic monocyclic or bicyclic system comprising 6 to 10 carbocyclic atoms, optionally substituted with 1 to 3 substituents, said substituents independently including halogen, hydroxyl, alkyl, alkenyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of aryl groups include phenyl and naphthyl, particularly phenyl.

[0242] In this disclosure, the term "heterocyclic group," alone or in combination, refers to a monocyclic or bicyclic system of 4 to 12 ring atoms (especially 4 to 9 ring atoms) that is monovalently saturated or partially unsaturated, comprising 1, 2, 3, or 4 heteroatoms including N, O, and S, with the remaining ring atoms being carbon, which is optionally substituted with 1 to 3 substituents, which independently include halogen, hydroxyl, alkyl, alkenyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of monocyclic saturated heterocyclic groups include azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, and tetrahydropyranyl. (rahydropyranyl), tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Examples of bicyclic saturated heterocyclic alkyl groups are 8-aza-bicyclo[3.2.1]octyl, quininecycloyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, or 3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated heterocyclic alkyl groups are dihydrofuranyl, imidazolinyl, dihydrooxazolyl, tetrahydropyridyl, or dihydropyranyl.

[0243] In this disclosure, the term "heteroaryl" alone or in combination refers to a monovalent aromatic heterocyclic monocyclic or bicyclic ring system of 5 to 12 ring atoms, comprising 1, 2, 3, or 4 heteroatoms including N, O, and S, with the remaining ring atoms being carbon, which is optionally substituted by 1 to 3 substituents, which independently include halogen, hydroxyl, alkyl, alkenyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of heteroaryl groups include pyrrole, furanyl, thiophene, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyrimidinyl, triazinyl, isoxazolyl, benzofuranyl, isothiazolyl, benzothiophene, indolyl, isindolyl, isobenzofuranyl, benzimidazolyl, benzooxazolyl, benzoisooxazolyl, benzothiazolyl, benzooxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxolinyl, carbazole, or acridineyl.

[0244] In this disclosure, the term "alicyclyl" refers to a cyclic system in which the rings are adipose. The cyclic system may comprise one or more rings, wherein at least one ring is adipose. Preferred alicycles comprise rings having about 5 to about 9 carbon atoms. Alicycles as used herein may optionally contain other substituents.

[0245] In this disclosure, the term "halogenated" in combination with another group indicates that the group is substituted with at least one halogen, particularly with one to five halogens, especially with one to four halogens, i.e., one, two, three, or four halogens. The "halogen" includes fluorine, chlorine, bromine, or iodine, particularly fluorine.

[0246] In this disclosure, the term "acyl" refers to a free radical having the general formula -C(O)-X, formed by removing a hydroxyl group from an organic acid, where X is typically an aliphatic, alicyclic, or aromatic group. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, and aliphatic phosphates. The acyl group used herein may optionally contain other substituents.

[0247] In this disclosure, the term "sulfonyl" alone or in combination refers to the -SO2 group.

[0248] In this disclosure, the term "formyl" alone or in combination means a -C(O)H group.

[0249] In this disclosure, the term "2'-O-methoxyethyl" (also known as 2'-MOE and 2'-OCH2CH2-OCH3 and MOE) refers to the O-methoxyethyl modification at the 2' position of the furanose ring.

[0250] In this disclosure, the term "cycloalkyl" refers, alone or in combination, to a cycloalkyl ring having 3 to 8 carbon atoms, particularly a cycloalkyl ring having 3 to 6 carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, more particularly cyclopropyl and cyclobutyl. A specific example of "cycloalkyl" is cyclopropyl.

[0251] In this disclosure, the term "gapmer" refers to an antisense oligonucleotide having a 5' wing-spacer-3' wing region, comprising a spacer region (gap region, central region) and regions flanking the central region (wing regions, 5' wing regions or 3' wing regions). The spacer region is either unmodified or contains at least one modification different from the modification of the wing region, including base modifications, internucleotide linker modifications, and ribose modifications. In some embodiments, the modifications in the 5' wing region and the 3' wing region are the same as or different from each other.

[0252] The term “GalNAc” has its conventional scientific meaning and here refers to N-acetylgalactosamine and its IUPAC name: 2-(acetylamino)-2-deoxy-D-galactose.

[0253] The term “GalNAc-decorated antisense oligonucleotide” has its conventional scientific meaning and here refers to an oligonucleotide used to interfere with gene transcription, wherein one or more GalNAc units are conjugated to an antisense oligonucleotide, for example, via at least one linker.

[0254] In this disclosure, the term "antigen-binding fragment" refers to a portion of a full-length antibody, wherein said portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain, or a VHH). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2 and Fv fragments, ScFv, and VHH.

[0255] In this disclosure, the terms “antibody-oligonucleotide conjugate” or “AOC” have their conventional scientific meaning and refer herein to any conjugate of an antibody (such as IgG, Fab, single-domain antibody, scFv, immunoglobulin, immunoglobulin fragment, one or more Vh domains, etc.) with any antisense oligonucleotide molecule that, when in contact with the cells of a subject (such as a human patient), can exert a therapeutic effect.

[0256] In this disclosure, the term "pharmaceutically acceptable carrier" refers to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, formulation adjuvants, or carriers conventionally used in the art for use with therapeutic agents, which together constitute a "pharmaceutical composition" for individual administration. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used. Suitable carriers are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymer micelles, or inorganic nanocarriers, etc.

[0257] In this disclosure, the renal tubule is one of the core structures of renal urinary function. The proximal tubule cell (PT) is the initial segment of the renal tubule and can be divided into the proximal convoluted tubule (PCT) and the proximal straight tubule (PST). Cells located in the proximal tubule are called proximal tubular cells, cells located in the proximal convoluted tubule are called proximal tubular cells, and cells located in the proximal straight tubule are called proximal straight tubular cells.

[0258] (II) Detailed Technical Solution

[0259] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0260] Table 1. Gapmer ASO tested in the examples

[0261] In this context, LNA- represents the connection between the 4' and 2' positions of the ribose as -CH2-O-, cET- represents the connection between the 4' and 2' positions of the ribose as -CH(CH3)-O-, i2MOEr represents the 2' position of the ribose as having -O-methoxyethyl modification (2'-OCH2CH2-OCH3), * represents the nucleotides being linked by a thiophosphate bond, # represents the nucleotides being linked by a methanesulfonylphosphatidyl ester bond, (5m) represents the 5' carbon in the cytosine base as having methylation modification, d represents the ribose as deoxyribose, and each nucleotide is separated by a " / ".

[0262] Table 2. Primer sequences used for qPCR

[0263] Example 1A: Comparison of the inhibitory effects of LNA-modified and MOE-modified gapmer ASO in C57BL / 6J mice.

[0264] Male C57BL / 6J mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. The housing conditions were controlled at room temperature of 20–25℃, relative humidity of 40%–70%, and alternating light and dark cycles of 12 hours. Feed was provided by Jiangsu Xietong Bioengineering Co., Ltd., feed production license: Su Si Zheng (2019) 01008. Drinking water was autoclaved tap water. Animals had free access to food and water. Cages and other equipment used for housing the animals were autoclaved before use. Mice were acclimatized for one week, and at 8 weeks of age, they were randomly divided into groups of 5 mice each, and administered a single subcutaneous dose of 1 μmol / kg. Two days after administration, the mice were euthanized with CO2, and kidneys were collected. Target gene expression was detected by RT-qPCR, and the qPCR results were normalized to the PBS group. The results are shown in the table below. The results showed that both LNA and MOE-modified Gapmer ASO exhibited significant inhibitory effects on target gene expression, and LNA-modified Gapmer ASO showed better inhibitory effects than MOE-modified Gapmer ASO.

[0265] Table 3. Inhibitory effect of ASO on mSGLT2

[0266] Example 1B: Activity assay of MsPA bond-modified ASO in C57BL / N mice

[0267] To investigate the effect of the MsPA-modified dual-targeting sequence ASO-92 on the activity of ASO-92 in mice, C57BL / 6N mice were administered a single subcutaneous injection of ASO-92 and ASO-92-04. Specific sequence information and modifications are shown in Table 1, where * represents a PS bond and # represents a MsPA bond. The dose was 0.3 μmol / kg. Seven days after administration, mice were euthanized, and kidneys were collected for RT-qPCR to detect SLGT2 expression. SLGT2 expression is shown in Table 4. The substitution of PS bonds with MsPA bonds did not affect the activity of ASO-92.

[0268] Table 4. Inhibitory effect of ASO on mSGLT2

[0269] Example 2: Testing of ASOs of different lengths for SGLT2 in C57BL / 6J mice

[0270] Male C57BL / 6J mice were housed in the same environment as in Example 1. After one week of acclimatization, the mice were randomly divided into groups of five at 8 weeks of age and administered a single subcutaneous dose of 0.3 μmol / kg. Six days after administration, the mice were euthanized with CO2, and kidneys were collected. Target gene expression was detected by RT-qPCR, and the qPCR results were normalized to the PBS group. The results are shown in the table below. The results showed that, under the same binding region and modification method, the effect on SGLT2 KD was 12mer>13mer>14mer>15mer>16mer.

[0271] Table 5. Inhibition effect of ASO of different lengths on mSGLT2

[0272] Example 3: Testing of shorter gapmer ASO in C57BL / 6J mice

[0273] Male C57BL / 6J mice were housed in the same environment as in Example 1. After one week of acclimatization, the mice were randomly divided into groups of four at 8 weeks of age. A single subcutaneous dose of Gapmer ASOs of varying lengths (10-12 meters) was administered. Fourteen days after administration, the mice were euthanized with CO2, and kidneys were harvested. Target gene expression was detected by RT-qPCR. The qPCR results were normalized to the PBS group. The results are shown in the table below. For 12-mer Gapmer ASOs, shorter sequences showed less inhibitory effect on the target mRNA.

[0274] Table 6. Inhibition effect of ASO of different lengths on mSGLT2

[0275] Example 4: Testing of ASOs for Gapmers of Different Lengths in URAT1 in C57BL / 6J Mice

[0276] Male C57BL / 6J mice were housed in the same environment as in Example 1. After one week of acclimatization, the mice were randomly divided into groups of five at 8 weeks of age. They were subcutaneously administered the drug at 3 μmol / kg on days 0 and 3. Six days after administration, the mice were euthanized with CO2, and kidneys were collected. Target gene expression was detected by RT-qPCR, and the qPCR results were normalized to the PBS group. The results are shown in the table below. The results showed that, under the same binding region and modification method, the URAT1 12mer Gapmer ASO exhibited the best KD effect.

[0277] Table 7. Inhibitory effect of ASO on mURAT1

[0278] Example 5: Efficacy test of Gapmer ASO targeting SGLT2 in diabetic model mice

[0279] Eight-week-old male db / db mice were acclimatized for one week, and blood glucose levels were measured by collecting blood from the ocular venous plexus after a 4-hour fast. Mice were randomly divided into groups of five based on blood glucose levels and body weight. The mice were administered subcutaneously at a dose of 1 μmol / kg once weekly for a total of four treatments. The first dose was recorded as Day 0. Fasting blood glucose was measured 48 hours after each administration, and monitoring continued after drug withdrawal. Results showed that LNA / cEt-modified 12mer Gapmer ASO exhibited the best hypoglycemic effect.

[0280] Table 8. Hypoglycemic effect of ASO in db / db mice

[0281] Example 6: Inhibitory effect of Gapmer ASO targeting MALAT1 on proximal renal tubular cells in C57BL / 6J mice.

[0282] Male C57BL / 6J mice were housed in the same environment as in Example 1. Mice were acclimatized for one week and randomly assigned to different groups at 8 weeks of age. Each mouse received a single subcutaneous dose of 1 μmol / kg. Three days after administration, the mice were euthanized with CO2, and their kidneys were collected. The collected kidneys were chopped, mixed evenly, and subjected to single-cell sequencing to detect gene expression levels and target gene inhibition in proximal tubular cells.

[0283] The results showed that the 12mer Gapmer ASO targeting MALAT1 could inhibit the target gene in proximal renal tubular cells (proximal convoluted tubules and proximal straight tubules).

[0284] Table 9. Inhibition of target genes in proximal renal tubule cells

Claims

1. An antisense oligonucleotide targeting proximal renal tubular cells, or a pharmaceutically acceptable salt thereof, said antisense oligonucleotide having a structure of a 5' wing region-spacer region-3' wing region, wherein, The 5' wing region consists of 1-5 nucleotides with the first chemical modification, and the 3' wing region consists of 1-5 nucleotides with the second chemical modification.

2. The antisense oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide is 10-18 nucleotides in length; Preferably, the antisense oligonucleotide is 12 or 13 nucleotides in length; Preferably, the 5' wing region consists of 2 or 3 nucleotides with a first chemical modification; Preferably, the 3' wing region consists of two or three nucleotides with a second chemical modification; Preferably, the antisense oligonucleotide has a 2-8-2 or 2-8-3 modification pattern, wherein, The first number represents the number of nucleotides in the 5' wing region, the second number represents the number of nucleotides in the spacer region, and the third number represents the number of nucleotides in the 3' wing region. Preferably, the first chemical modification involves attaching one or more first linking groups between the 4' and 2' positions of the ribose; Preferably, the second chemical modification involves attaching one or more second linking groups between the 4' and 2' positions of the ribose; Preferably, the first linking group and the second linking group each independently comprise -[C(R1)(R2)] n -, -C(R1)=C(R2)-, -C(R1)=N-, -C(=NR1)-, -C(=O)-, -C(=S)-, -O-, -Si(R1)2-, -S(=O) x -、-N(R1)-、-R-[C(R1)(R2)]n-R'-、-[C(R1)(R2)]nR-R'- or -R-R'-[C(R1)(R2)]n-、-RC(R1)=C(R2)-R'-、-R-R'-C(R1)=C(R2)-、-C(R1)=C(R2)-R-R'-;wherein, R and R', each independently, include single bonds, -N(R3)-、-O-、-S-、-Se-、-Si(R4)(R5)-、-C(=O)-、-C(=S)-、-C(=NR3)-; Where x is 0, 1, or 2; n is 1, 2, 3 or 4; R1, R2, R3, R4, and R5, each independently, include H, a protecting group, a hydroxyl group, and substituted or unsubstituted C1-C. 12 Alkyl, substituted or unsubstituted C 2- C 12 alkenyl, substituted or unsubstituted C2-C 12 Alkyne, substituted or unsubstituted C5-C 20 Aryl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroaryl, substituted or unsubstituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-J1), substituted acyl, CN, sulfonyl (S(=O)2-J1), sulfonyloxy (S(=O)-J1) or -C(=NH)-NH2, or R1 and R2 connected to form C3-C6 cycloalkyl or heterocyclic alkyl; J1 and J2, each independently, include H, substituted or unsubstituted C1-C. 12 Alkyl, substituted or unsubstituted C2-C 12 alkenyl, substituted or unsubstituted C2-C 12 Alkyne, substituted or unsubstituted C5-C 20 Aryl, substituted or unsubstituted acyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted C1-C 12 Aminoalkyl or protecting group; Preferably, the first linking group and the second linking group each independently include -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -[C(R1)(R2)]nN(R3)-, [C(R1)(R2)]nN(R3)-O-, -[C(R1)(R2)]nON(R3)-, -[C(R1)(R2)]nS-, -C(R1)=C(R2)-, -C(R1)=N-; wherein R1, R2, and R3 each independently are H, a protecting group, or C1-C 12 alkyl; Preferably, the first and second linking groups each independently include -(CH2)3-, -(CH2)2-, -CH2-O-, -(CH2)2-O-, -CH2-ON(R1)-, -CH2-N(R1)-O-, -CH(CH3)-O-, -C(C2H4)O-, -CH2-N(R1)-, -CH(R1)-NH-, -CH(R1)-N(R1)-, CH2-S-, -CH(CH3)-S-, or -C(=O)-N(R1)-, wherein each R1 is independently H, a protecting group, or C1-C. 12 alkyl; Preferably, the first linking group and the second linking group are each independently -CH2-O- or -CH(CH3)-O-; Preferably, when the first or second linker is -CH2-O-, the nucleotide connecting the first or second linker between the 4' and 2' positions of the ribose is an LNA nucleotide, which has the following structure: Where Bx refers to a base; Preferably, when the first or second linker is -CH(CH3)-O-, the nucleotide linking the first or second linker between the 4' and 2' positions of the ribose is a restricted ethyl-modified nucleotide having the following structure: Where Bx refers to a base; Preferably, the first linking group and the second linking group are the same or different, and the first chemical modification and the second chemical modification are the same or different; Preferably, the first linking group and the second linking group are the same, and the first chemical modification and the second chemical modification are the same; Preferably, the first linking group is -CH2-O-, and the second linking group is -CH2-O-; Preferably, the first linking group is -CH(CH3)-O-, and the second linking group is -CH(CH3)-O-; Preferably, the first chemical modification involves linking -CH2-O- between the 4' and 2' positions of the ribose; Preferably, the second chemical modification involves linking -CH2-O- between the 4' and 2' positions of the ribose; Preferably, the first chemical modification is the attachment of -CH(CH3)-O- between the 4' and 2' positions of the ribose; Preferably, the second chemical modification is the attachment of -CH(CH3)-O- between the 4' and 2' positions of the ribose.

3. The antisense oligonucleotide of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide further has base modifications, ribose modifications, and / or phosphate backbone modifications; Preferably, the base modification includes methylation modification at any site of purine or pyrimidine in any nucleotide; Preferably, the base modification includes methylation modification of the 5' position of cytosine in any nucleotide; Preferably, the phosphate skeleton modification includes thiophosphate modification, methylated phosphate modification, dimercaptophosphate modification, or 5'-(E)-VP modification; Preferably, the phosphate backbone modification is thiophosphate modification; Preferably, one or more of the antisense oligonucleotides have a nucleotide internucleotide bond modified with thiophosphate; Preferably, the nucleotides in the spacer region have deoxyribose and / or ribose; Preferably, one or more nucleotides in the spacer region have deoxyribose; Preferably, the bases of the nucleotides in the antisense oligonucleotide are selected from any one or more of the following: adenine, thymine, guanine, and 5-methylcytosine.

4. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3, wherein the antisense oligonucleotide targets a target gene; Preferably, the antisense oligonucleotide is at least partially complementary to any nucleotide segment in the transcribed mRNA of the target gene; Preferably, the antisense oligonucleotide is completely complementary to any segment of nucleotides in the transcribed mRNA of the target gene; Preferably, the target gene includes SGLT2, URAT1, or MALAT1; Preferably, the base sequence of the antisense oligonucleotide comprises a base sequence that differs from the base sequence of the nucleotides shown in any one of SEQ ID NO. 15, 16, 19, 20, 24, 25, 26, 28 by no more than 4, 3, 2, or 1 bases. Preferably, the base sequence of the antisense oligonucleotide comprises the base sequence of the nucleotide shown in any one of SEQ ID NO. 15, 16, 19, 20, 24, 25, 26, 28, or the base sequence of the antisense oligonucleotide is as shown in any one of SEQ ID NO. 15, 16, 19, 20, 24, 25, 26, 28; Preferably, the nucleotides of the antisense oligonucleotide are linked by phosphate thioester bonds and / or methanesulfonylphosphatidyl ester linkages; Preferably, the antisense oligonucleotide is selected from any one of the following: (1) Having the base sequence of the nucleotides shown in SEQ ID NO.15 and the modification pattern shown in 2-8-2; (2) Having the base sequence of the nucleotides shown in SEQ ID NO.16 and the modification pattern shown in 2-8-2; (3) Having the base sequence of the nucleotides shown in SEQ ID NO.19 and the modification pattern shown in 2-8-3; (4) Having the base sequence of the nucleotides shown in SEQ ID NO.20 and the modification pattern shown in 2-8-2; (5) Having the base sequence of the nucleotides shown in SEQ ID NO.24 and the modification pattern shown in 2-8-3; (6) Having the base sequence of the nucleotides shown in SEQ ID NO.25 and the modification pattern shown in 2-8-2; (7) Having the base sequence of the nucleotides shown in SEQ ID NO.26 and the modification pattern shown in 2-8-2; or (8) Having the base sequence of the nucleotides shown in SEQ ID NO.28 and the modification pattern shown in 2-8-2; in, The first number in the modification pattern represents the number of nucleotides in the 5' wing region, the second number represents the number of nucleotides in the spacer region, and the third number represents the number of nucleotides in the 3' wing region.

5. The antisense oligonucleotide as described in any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide is selected from any one of the following: (1)LNA-G* / LNA-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / LNA-T* / LNA-(5m)C(SEQ ID NO.1), (2)LNA-A* / LNA-G* / dA* / dT* / d(5m)C* / dT* / dT* / dG* / dG* / dT* / LNA-G* / LNA-A(SEQ ID NO.6), (3)LNA-A* / LNA-G* / dA* / dA* / dT* / dG* / d(5m)C* / dG* / dG* / d(5m)C* / LNA-T* / LNA-G(SEQ ID NO.11), (4)LNA-(5m)C* / LNA-(5m)C* / dA* / dG* / dG* / dA* / dG* / dT* / dT* / d(5m)C* / LNA-A* / LNA-G (SEQ ID NO. 12), (5)LNA-A* / LNA-G* / dT* / dT* / d(5m)C* / dA* / d(5m)C* / dT* / dG* / dA* / LNA-A* / LNA-T (SEQ ID NO.14), (6)cET-G* / cET-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / cET-T* / cET-(5m)C(SEQ ID NO.2), (7)LNA-A* / LNA-G* / dA* / dT* / d(5m)C* / dT* / dT* / dG* / dG* / dT* / LNA-G* / LNA-A* / LNA-A (SEQ ID NO.5), (8)LNA-A* / LNA-G* / dA* / dA* / dT* / dG* / d(5m)C* / dG* / dG* / d(5m)C* / LNA-T* / LNA-G* / LNA-A (SEQ ID NO. 10), in, LNA- represents the connection between the 4' and 2' positions of the ribose -CH2-O-, cET- represents the connection between the 4' and 2' positions of the ribose -CH(CH3)-O-, * represents the connection between nucleotides via a thiophosphate bond, (5m) indicates that the 5' carbon in the cytosine base is methylated, d represents that the ribose is deoxyribose, and each nucleotide is separated by " / ". Preferably, the antisense oligonucleotide is 12 nucleotides in length; Preferably, the antisense oligonucleotide has a 2-8-2 modification pattern, wherein the first number in the modification pattern represents the number of nucleotides in the 5' wing region, the second number represents the number of nucleotides in the spacer region, and the third number represents the number of nucleotides in the 3' wing region. Preferably, the antisense oligonucleotide is selected from any one of the following: (1) Having the base sequence of the nucleotides shown in SEQ ID NO.15 and the modification pattern shown in 2-8-2; (2) Having the base sequence of the nucleotides shown in SEQ ID NO.16 and the modification pattern shown in 2-8-2; (3) Having the base sequence of the nucleotides shown in SEQ ID NO.20 and the modification pattern shown in 2-8-2; (4) Having the base sequence of the nucleotides shown in SEQ ID NO.25 and the modification pattern shown in 2-8-2; (5) Having the base sequence of the nucleotides shown in SEQ ID NO.26 and the modification pattern shown in 2-8-2; or (6) Having the base sequence of the nucleotides shown in SEQ ID NO.28 and the modification pattern shown in 2-8-2; In the modification pattern, the first number represents the number of nucleotides in the 5' wing region, the second number represents the number of nucleotides in the spacer region, and the third number represents the number of nucleotides in the 3' wing region. Preferably, the antisense oligonucleotide is selected from any one of the following: (1)LNA-G* / LNA-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / LNA-T* / LNA-(5m)C(SEQ ID NO.1), (2)cET-G* / cET-G* / d(5m)C* / dA* / dT* / dG* / dA* / dG* / d(5m)C* / dT* / cET-T* / cET-(5m)C(SEQ ID NO.2), (3)LNA-A* / LNA-G* / dA* / dT* / d(5m)C* / dT* / dT* / dG* / dG* / dT* / LNA-G* / LNA-A (SEQ ID NO. 6), (4)LNA-A* / LNA-G* / dA* / dA* / dT* / dG* / d(5m)C* / dG* / dG* / d(5m)C* / LNA-T* / LNA-G(SEQ ID NO.11), (5)LNA-(5m)C* / LNA-(5m)C* / dA* / dG* / dG* / dA* / dG* / dT* / dT* / d(5m)C* / LNA-A* / LNA-G(SEQ ID NO.12), or (6)LNA-A* / LNA-G* / dT* / dT* / d(5m)C* / dA* / d(5m)C* / dT* / dG* / dA* / LNA-A* / LNA-T (SEQ ID NO. 14), In this context, LNA- represents the connection between the 4' and 2' positions of the ribose, -CH2-O-; cET- represents the connection between the 4' and 2' positions of the ribose, -CH(CH3)-O-; * represents the connection between nucleotides via a thiophosphate bond; (5m) indicates that the 5' carbon in the cytosine base is methylated; d represents that the ribose is deoxyribose; and each nucleotide is separated by " / ".

6. An antisense oligonucleotide conjugate comprising the antisense oligonucleotide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof; Preferably, the antisense oligonucleotide conjugate further comprises a targeting ligand, wherein the targeting ligand is conjugated to the antisense oligonucleotide; Preferably, the targeting ligand is conjugated to the 3' end and / or 5' end of the antisense oligonucleotide; Preferably, the targeting ligand comprises a protein, a small molecule compound, a carbohydrate, or a lipid; Preferably, the protein includes natural proteins, synthetic polyamino acids, polypeptides, and antibodies; Preferably, the natural protein includes human serum albumin, low-density lipoprotein, or globulin; Preferably, the synthesized polyamino acids include polylysine, poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactic acid-co-ethylene glycol) copolymer, diethylene ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer, polyethylene glycol, polyvinyl alcohol, polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphoric acid. Preferably, the carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, amino sugars, or hyaluronic acid; Preferably, the lipids include fatty acids, sterols, or phospholipids; Preferably, the fatty acids include capric acid, caprylic acid, lauric acid, palmitic acid, myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, or eicosenoic acid. Preferably, the sterols include cholesterol, cholesterol, stigmasterol, cholesterol acid, or ergosterol; Preferably, the phospholipid comprises di-hexadecyl-racemic glycerol or triethylamine 1,2-di-O-hexadecyl-racemic glycerol-3-hydrophosphonate; Preferably, the targeting ligand comprises amino sugars; Preferably, the targeting ligand comprises an N-acetylgalactosamine moiety; Preferably, the GalNac portion is a monovalent GalNAc portion, a divalent GalNAc portion, a trivalent GalNAc portion, or a tetravalent GalNAc portion; Preferably, the targeting ligand has the following structure:

7. A composition comprising the antisense oligonucleotide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, or the composition comprising the antisense oligonucleotide conjugate of claim 6; Preferably, the composition further contains a pharmaceutically acceptable carrier; Preferably, the composition further comprises one or more additional therapeutic components; Preferably, the composition is packaged in a box, container, packaging material, dispenser, pre-filled syringe, or vial.

8. A cell containing the antisense oligonucleotide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, the antisense oligonucleotide conjugate of claim 6, or the composition of claim 7.

9. Any of the following applications of the antisense oligonucleotide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, the antisense oligonucleotide conjugate of claim 6, the composition of claim 7, or the cell of claim 8: (1) Use in the preparation of medicines for the prevention and / or treatment of diseases. (2) Application in suppressing target gene expression (3) Application in targeting proximal renal tubules; Preferably, the disease includes diseases caused by abnormal expression of any one or more of the following genes: SGLT2, URAT1, or MALAT1; Preferably, the inhibition of target gene expression is the inhibition of target gene expression in proximal renal tubular cells; Preferably, the target gene is a gene expressed in proximal renal tubular cells; Preferably, the target gene includes one or more of SGLT2, URAT1, or MALAT1; Preferably, the proximal renal tubule includes a proximal convoluted tubule and / or a proximal straight tubule; Preferably, the proximal renal tubular cells include proximal convoluted tubular cells and / or proximal straight tubular cells.

10. A method for reducing the expression of a target gene, the method comprising contacting the antisense oligonucleotide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, the antisense oligonucleotide conjugate of claim 6, the composition of claim 7, or the cell of claim 8 with the target gene; Preferably, the method involves reducing the expression of the target gene in proximal renal tubular cells; Preferably, the method is for non-therapeutic purposes; Preferably, the method is performed in vitro; Preferably, the target gene is a gene expressed in proximal renal tubular cells; Preferably, the target gene includes one or more of SGLT2, URAT1, or MALAT1; Preferably, the proximal renal tubular cells include proximal convoluted tubular cells and / or proximal straight tubular cells.

11. The use of the antisense oligonucleotide of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, the antisense oligonucleotide conjugate of claim 6, the composition of claim 7, or the cell of claim 8 in the prevention and / or treatment of diseases; Preferably, the disease includes diseases caused by abnormal expression of any one or more of the following genes: SGLT2, URAT1, or MALAT1; Preferably, the inhibition of target gene expression is the inhibition of target gene expression in proximal renal tubular cells; Preferably, the target gene is a gene expressed in proximal renal tubular cells; Preferably, the target gene includes one or more of SGLT2, URAT1, or MALAT1; Preferably, the proximal renal tubule includes a proximal convoluted tubule and / or a proximal straight tubule; Preferably, the proximal renal tubular cells include proximal convoluted tubular cells and / or proximal straight tubular cells.

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