Aptamer conjugate for targeted degradation of sclerostin

Abstract

The present invention relates to an aptamer conjugate for targeted degradation of sclerostin. Specifically, the present invention relates to a conjugate of an aptamer against sclerostin and a ligand capable of targeting a protein degradation pathway, and use of the conjugate in degrading sclerostin.

Description

Aptamer conjugates for targeted degradation of sclerostin Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to aptamer conjugates for the targeted degradation of sclerostin. More specifically, the present invention relates to conjugates of aptamers targeting sclerostin and ligands capable of targeting protein degradation pathways, as well as the use of such conjugates for degrading sclerostin.

[0002] Background of the Invention

[0003] At present, targeted protein degradation (TPD) has become a very promising targeted therapy technology, which is mainly based on two protein degradation pathways: the proteasome pathway and the lysosome pathway. For the proteasome pathway, bifunctional molecules that can connect target proteins and E3 enzymes are called protein hydrolysis targeting chimeras (PROTACs), which use the ubiquitin-proteasome system to degrade intracellular target proteins through a series of enzymes. For the lysosomal pathway, the bifunctional molecules that can connect target proteins and transmembrane receptors are the most well-known lysosomal targeting chimeras (LYTACs), which mediate the internalization of target proteins into lysosomes after endocytosis. The lysosomal microenvironment then degrades the target transmembrane or extracellular proteins, which is a good supplement to PROTAC.

[0004] Aptamers are short fragments of DNA or RNA that can recognize and bind to target molecules (usually proteins) through their specific 3D structures. These fragments are selected from randomly synthesized oligodeoxynucleotides or oligonucleotide libraries using SELEX (systematic evolution of ligands by exponential enrichment). Compared to small molecule binders, aptamers often have better selectivity and higher affinity. Compared to antibodies, aptamers have a simpler evolution process, are easy to modify, have affinity adjustments, have low immunogenicity, and are versatile in structural design and engineering, making them ideal recognition ligands for their targets. As a result, an increasing number of aptamers are being developed and used as therapeutic agents and probes.

[0005] Sclerostin is a promising target for osteoporosis drug development (Rey and Ellies, 2010). Humanized monoclonal antibodies against human sclerostin have been reported to promote bone formation and increase bone mass in clinical trials with good tolerability. However, therapeutic antibodies have several major challenges, including high immunogenicity (Padhi, Jang et al., 2011; Padhi, Allison et al., 2014), high production costs (Baker, 2015; Bradbury and Pluckthun, 2015; Groff, Brown et al., 2015), and instability requiring continuous cold chain transportation and storage (Jayasena, 1999). Furthermore, sclerostin antibodies may not be effective against intracellular sclerostin. Therefore, for sclerostin-targeted therapeutics, alternative anti-sclerostin agents that are non-immunogenic, easy to produce, low-cost, highly stable, and capable of targeting intracellular sclerostin are desired.

[0006] Summary of the Invention

[0007] The present invention provides at least the following embodiments:

[0008] Embodiment 1. An aptamer conjugate comprising i) an aptamer that specifically binds to sclerostin, and ii) a specific ligand for E3 ubiquitin ligase (E3) and / or a specific ligand for asialoglycoprotein receptor (ASGPR).

[0009] Embodiment 2. The aptamer conjugate of embodiment 1, wherein the aptamer that specifically binds to sclerostin comprises

[0010] i) a nucleotide sequence that is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to any one of SEQ ID NOs: 1-17; or

[0011] ii) at least 30, at least 35, at least 40, at least 45, at least 50 or more contiguous nucleotides of any one of SEQ ID NOs: 1-17; or

[0012] iii) the nucleotide sequence of any one of SEQ ID NOs: 1-17, preferably, the nucleotide sequence of SEQ ID NO: 17.

[0013] Embodiment 3. The aptamer conjugate of embodiment 1 or 2, wherein the aptamer that specifically binds to sclerostin has a Kd (dissociation constant) for sclerostin of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM or less.

[0014] Embodiment 4. The aptamer conjugate of any one of Embodiments 1-3, wherein the aptamer that specifically binds to sclerostin is a modified aptamer, and the modified aptamer may contain one or more modifications that impart enhanced nuclease resistance to the aptamer and / or modifications that extend the in vivo half-life of the aptamer.

[0015] Embodiment 5. The aptamer conjugate of embodiment 4, wherein the modification comprises a 3' inverted deoxythymidine (3'idT) modification.

[0016] Embodiment 6. The aptamer conjugate of embodiment 4, wherein the modification comprises replacing one or more naturally occurring nucleotides with modified nucleotides, wherein the modified nucleotides are selected from 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and / or 2'propyleneoxy modified nucleotides, preferably 2'-methoxy modified nucleotides.

[0017] Embodiment 7. The aptamer conjugate of embodiment 4, wherein the modification comprises an internucleotide modification, such as an internucleotide phosphorothioate linkage modification.

[0018] Embodiment 8. The aptamer conjugate of embodiment 4, wherein the aptamer comprises a 2'-methoxy (2'-OMe) modification and / or a 3' inverted deoxythymidine (3'idT) modification.

[0019] Embodiment 9. The aptamer conjugate of any one of embodiments 1-8, wherein the aptamer is further conjugated to a fatty acid.

[0020] Embodiment 10. The aptamer conjugate of embodiment 9, wherein the fatty acid is selected from palmitic acid (PA), dodecanedioic acid (DA), tetradecanedioic acid, hexadecanedioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA), preferably, the fatty acid is octadecanedioic acid.

[0021] Embodiment 11. The aptamer conjugate of any one of embodiments 1-10, wherein the E3-specific ligand is selected from von Hippel-Landau (VHL), murine double minute 2 (MDM2), and cereblon (CRBN); preferably, the E3-specific ligand is VHL, for example, the VHL comprises a structure shown in the following formula:

[0022] Embodiment 12. The aptamer conjugate of any one of embodiments 1-11, wherein the E3-specific ligand is conjugated to the aptamer via a linker, for example, to the 5' end of the aptamer.

[0023] Embodiment 13. The aptamer conjugate of embodiment 12, wherein the linker is a bifunctional linker,

[0024] For example, the bifunctional linker comprises one of the following structures:

[0025] ;or

[0026] The bifunctional linker comprises the following structure:

[0027] Embodiment 14. The aptamer conjugate of embodiment 13, wherein the aptamer conjugate comprises the following structure: (FA / E3 ligand)-bifunctional linker-aptamer nucleotide sequence (5'-3'), wherein FA represents fatty acid, E3 ligand represents E3-specific ligand, wherein FA and E3 ligand are conjugated to the 5' end of the aptamer nucleotide sequence via a bifunctional linker, preferably, the fatty acid is octadecanedioic acid and / or the E3-specific ligand is VHL.

[0028] Embodiment 15. The aptamer conjugate of embodiment 14, wherein the aptamer conjugate comprises a structure represented by the following formula:

[0029] Embodiment 16. The aptamer conjugate of any one of embodiments 1-10, wherein the specific ligand of the asialoglycoprotein receptor (ASGPR) is N-acetylgalactosamine (GalNAc).

[0030] Embodiment 17. The aptamer conjugate of any one of embodiments 1-16, wherein the ASGPR-specific ligand, such as GalNAc, is conjugated to the aptamer via a linker.

[0031] Embodiment 18. The aptamer conjugate of embodiment 16 or 17, wherein the aptamer conjugate comprises a structure represented by the following formula:

[0032] in

[0033] Embodiment 19. The aptamer conjugate of any one of embodiments 16-18, wherein the aptamer conjugate comprises a structure represented by the following formula:

[0034] Embodiment 20. The aptamer conjugate of any one of embodiments 1-19, wherein the aptamer nucleotide sequence (5'-3' direction) is

[0035] C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT, where (OMe) represents the 2'-methoxy (2'-OMe) modification of the corresponding nucleotide and idT represents the 3' inverted deoxythymidine modification.

[0036] Embodiment 21. The aptamer conjugate according to any one of embodiments 1-20, wherein the aptamer conjugate is used for targeted degradation of sclerostin.

[0037] Embodiment 22. The aptamer conjugate of embodiment 21, wherein the aptamer conjugate is used for targeted degradation of sclerostin in cells.

[0038] Embodiment 23. The aptamer conjugate of embodiment 22, wherein the cell is a cancer cell, for example, the cancer cell is a cancer cell that highly expresses sclerostin, or the cancer cell is a cancer cell that highly expresses sclerostin and asialoglycoprotein receptor (ASGPR).

[0039] Embodiment 24. The aptamer conjugate of embodiment 23, wherein the cancer cell is a breast cancer cell (preferably a triple-negative breast cancer cell) or a liver cancer cell.

[0040] Embodiment 25. A method of treating a sclerostin-related disease, comprising administering a therapeutically effective amount of the aptamer conjugate of any one of embodiments 1-20 to a subject in need thereof, such as a human.

[0041] Embodiment 26. The method of embodiment 25, wherein the sclerostin-related disease is selected from osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis, rheumatoid arthritis, bone fracture, osteoarthritis, myeloma, hypophosphatemic rickets, liver cancer and triple-negative breast cancer.

[0042] Embodiment 27. A pharmaceutical composition comprising at least one aptamer conjugate according to any one of embodiments 1-20, and a pharmaceutically acceptable carrier or excipient.

[0043] Embodiment 28. Use of the aptamer conjugate according to any one of embodiments 1 to 20 or the pharmaceutical composition according to embodiment 27 in the preparation of a medicament, wherein the medicament is for treating a sclerostin-related disease.

[0044] Embodiment 29. The use according to embodiment 28, wherein the sclerostin-related disease is selected from osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis, rheumatoid arthritis, bone fracture, osteoarthritis, myeloma, hypophosphatemic rickets, liver cancer and triple-negative breast cancer.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1. Synthesis of OA-conjugated sclerostin aptamer-linked VHL ligand. (A) Synthesis route of the VHL E3 ligand linked to the OA-conjugated sclerostin aptamer. (B) Purification of Apc001OA-VHL by HPLC. (C) ESI-MS analysis of Apc001OA-VHL.

[0047] Figure 2. VHL E3 ligand linked to OA-conjugated sclerostin aptamer can significantly degrade intracellular sclerostin in MDA-MB-231 cells in vitro. MDA-MB-231 cells were treated with 0.7 μM Apc001OA-VHL, 1.4 μM Apc001OA-VHL, and PBS, respectively.

[0048] Figure 3. The degradation of intracellular sclerostin by VHL E3 ligand linked to OA-conjugated sclerostin aptamer can be sustained for 24 hours in MDA-MB-231 cells in vitro. MDA-MB-231 cells were treated with 0.7 μM Apc001OA-VHL or PBS.

[0049] Figure 4. VHL E3 ligand linked to OA-conjugated sclerostin aptamer can inhibit cell proliferation of MDA-MB-231 cells in vitro. MDA-MB-231 cells were treated with 700 nM Apc001OA-VHL, Apc001OA, or PBS.

[0050] Figure 5. VHL E3 ligand linked to OA-conjugated sclerostin aptamer can inhibit cell migration in MDA-MB-231 cells in vitro. MDA-MB-231 cells were treated with 700 nM Apc001OA-VHL, Apc001OA, or PBS.

[0051] Figure 6. VHL E3 ligand linked to an OA-conjugated sclerostin aptamer inhibits MDA-MB-231 cell viability in vitro. MDA-MB-231 cells were treated with 700 nM Apc001OA-VHL, Apc001OA, or PBS. Statistical data are presented as mean ± SD using unpaired t-tests.

[0052] FIG7 . VHL E3 ligand linked to OA-conjugated sclerostin aptamer can inhibit tumor growth in a subcutaneous mouse model inoculated with MDA-MB-231 cells and tumor metastasis in an orthotopic mouse model inoculated with 4T1 cells.

[0053] Figure 8. Construction of GalNAc-Apc001 conjugate.

[0054] Figure 9. GalNAc mediates cell-specific internalization and trafficking of FAM-labeled Apc001 and Scramble sequences to lysosomes in ASGPR+ cells. (a, b) Confocal microscopy images of HepG2 cells (a) and A375 cells (b) incubated with 1 μM GalNAc-Apc001-FAM, GalNAc-Scramble-FAM, and Apc001-FAM, respectively, for 2 hours. Cell nuclei were stained with Hoechst 33342 (blue); FAM (green); and lysosomes were stained with Lysotracker Red. Scale bar, 10 μm.

[0055] Figure 10. GalNAc-Apc001 mediates sclerostin degradation. Western blot analysis was performed to analyze the temporal accumulation (0-8 hours) and degradation (8-18 hours) of sclerostin transported by GalNAc-Apc001 into HepG2 cells. HepG2 cells were incubated with DMEM medium containing a 1 μM GalNAc-Apc001 / Sclerostin (1:1) mixture for 0-8 hours. At the 8-hour time point, the medium was replaced with standard DMEM medium. GAPDH was used as an internal control.

[0056] Figure 11. GalNAc-Apc001 can significantly attenuate the inhibitory effect of sclerostin on Wnt signaling in vitro using additional ASGPR+ cells. Effects of GalNAc-Apc001, Apc001, and sclerostin antibody (Scl. Antibody) on sclerostin-induced Wnt signaling in HEK293 cells in the presence of HepG2 cells or A375 cells, respectively. Differences between groups were determined using a one-way ANOVA with Tukey's test. n = 3 per group, *P < 0.05, **P < 0.01, ***P < 0.001. Note: PBS represents the group treated with 1× PBS, with the same volume as the other groups. Scl. represents the group treated with 100 nM sclerostin. Wnt represents the group transfected with the Wnt-1 plasmid.

[0057] Figure 12. GalNAc-conjugated favorably modified aptamers promote bone anabolism in osteogenesis imperfecta mice (Col1a2+ / G610C). Bar graphs showing the structural parameters TB.conn.D, TB.vBMD, TB.BV / TV, TB.N, TB.Th, and TB.Sp from ex vivo microCT of distal femoral trabecular bone. Note: TB.conn.D: trabecular connection density; TB.vBMD: trabecular volume mineral density; TB.BV / TV: trabecular bone volume fraction; TB.N: trabecular number; TB.Th: trabecular thickness; TB.Sp: trabecular separation. Data were normalized by the parameters in the OI baseline group and expressed as mean ± SD, followed by one-way ANOVA analysis with Tukey's post hoc test. N = 6-12 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 compared with OI-Veh.

[0058] Detailed Description of the Invention

[0059] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual"; Lewin, "Genes VIII"; and Roitt et al., "Immunology" (8th edition), as well as to the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se, which will be understood by those skilled in the art. Reference is also made, for example, to standard manuals, the above-mentioned general prior art and other references cited therein.

[0060] definition

[0061] As used herein, the term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide, or a modified form thereof and analogs thereof. Nucleotides include species including purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs) and pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogs).

[0062] As used herein, "nucleic acid," "oligonucleotide," and "polynucleotide" are used interchangeably to refer to polymers of nucleotides and include DNA, RNA, DNA / RNA hybrids, and modifications of these types of nucleic acids, oligonucleotides, and polynucleotides, including the addition of various entities or moieties at any position of the nucleotide unit. The terms "polynucleotide," "oligonucleotide," and "nucleic acid" include double- and single-stranded molecules. Nucleic acid, oligonucleotide, and polynucleotide are broader terms than the term aptamer, and thus the terms nucleic acid, oligonucleotide, and polynucleotide include aptamers but are not limited to aptamers.

[0063] As used herein, "aptamer" refers to a non-naturally occurring nucleic acid that has a desired effect on a target molecule. The desired effect includes, but is not limited to, binding to the target, catalytically changing the target, reacting with the target in a manner that modifies or changes the target or the functional activity of the target, covalently linking the target, and promoting reactions between the target and other molecules. In some embodiments, the effect is specific binding affinity for a target molecule (e.g., sclerostin), such a target molecule is a three-dimensional chemical structure rather than a polynucleotide that binds to the aptamer by a mechanism that is independent of Watson / Crick base pairing or triple helix formation, wherein the aptamer is not a nucleic acid with a known physiological function of being bound by the target molecule. In this context, "specific binding" of an aptamer to its target (e.g., sclerostin) means that the aptamer generally binds to its target with a much higher affinity than it binds to other non-target components in a mixture or sample. An aptamer can be a single-stranded DNA, a single-stranded DNA, a single-stranded DNA / RNA hybrid, or a double-stranded DNA molecule.

[0064] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)). Many algorithms can be used to determine percent sequence identity. An example of an algorithm suitable for determining percent sequence identity is the algorithm used in the Basic Local Alignment Search Tool (hereinafter "BLAST"), see, for example, Altschul et al., J. Mol. Biol. 215: 403-410, 1990 and Altschul et al., Nucleic Acids Res., 15: 3389-3402, 1997. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (hereinafter "NCBI"). Default parameters used in determining sequence identity using software available from NCBI (such as BLASTN for nucleic acid sequences) are described in McGinnis et al. Nucleic Acids Res., 32: W20-W25, 2004.

[0065] Aptamer conjugates

[0066] In one aspect, the present invention provides an aptamer conjugate comprising i) an aptamer that specifically binds to sclerostin, and ii) a specific ligand for E3 ubiquitin ligase (E3) and / or a specific ligand for asialoglycoprotein receptor (ASGPR).

[0067] The sclerostin described herein is preferably human sclerostin.

[0068] An exemplary human sclerostin comprises the following amino acid sequence:

[0069] In some embodiments, the aptamer comprises a nucleotide sequence that is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to any one of SEQ ID NOs: 1-17, or the aptamer comprises at least 30, at least 35, at least 40, at least 45, at least 50, or more contiguous nucleotides of any one of SEQ ID NOs: 1-17. In some embodiments, the aptamer specifically binds to sclerostin. In some preferred embodiments, the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs: 1-17, more preferably, the aptamer comprises the nucleotide sequence of SEQ ID NO: 17.

[0070] In some embodiments, the aptamer or aptamer conjugate of the present invention has a Kd (dissociation constant) for sclerostin of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM or less. The Kd is determined, for example, by enzyme-linked oligonucleotide assay (ELONA).

[0071] In some embodiments, the aptamer of the present invention can be a modified aptamer, and the modified aptamer can include one or more modifications. For example, the modification is a modification that confers enhanced nuclease resistance to the aptamer and / or a modification that extends the in vivo half-life of the aptamer.

[0072] The modification includes, for example, 3' and / or 5' modification, such as 3' and 5' capping. In some embodiments, the nucleic acid molecule is capped at the 3' end with inverted deoxythymidine, ie, 3' inverted deoxythymidine (3'idT) modification.

[0073] The modification can also include replacing one or more naturally occurring nucleotides with modified nucleotides. For example, the modified nucleotides include but are not limited to nucleotides modified with 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and / or 2'propyleneoxy (i.e., the hydroxyl group at the 2' position of the ribose is replaced by fluoro, methoxyethyl, methoxy or propyleneoxy). The modified nucleotides can also include C-5 modified pyrimidines. The term "C-5 modified pyrimidine" refers to a pyrimidine with a modification at the C-5 position. C-5 modified pyrimidines can enhance the nuclease resistance of oligonucleotides and are known in the art, for example, as described in International Patent Application WO 2011 / 130195 and the literature cited therein. In some preferred embodiments, the modification is a 2'-methoxy (2'-OMe) modification. In some embodiments, one or more, for example, four nucleotides at the 5' and / or 3' ends of the nucleic acid molecule are modified, for example, with a 2'-methoxy (2'-OMe) modification.

[0074] The modifications also include internucleotide modifications, such as internucleotide modifications with uncharged bonds (such as methylphosphonate, phosphotriester, phosphoamine, carbamate, etc.) and internucleotide modifications with charged bonds (such as phosphorothioate, dithiophosphate, etc.), internucleotide modifications with intercalators (such as acridine, psoralen, etc.), internucleotide modifications containing chelators (such as metals, radioactive metals, boron, oxidative metals, etc.), internucleotide modifications containing alkylating agents and internucleotide modifications with modified bonds (such as alpha anomeric nucleic acids, etc.).

[0075] In some embodiments, the aptamer may comprise a combination of the above modifications. For example, the aptamer may comprise a 2'-methoxy (2'-OMe) modification and / or a 3' inverted deoxythymidine (3'idT) modification.

[0076] In some embodiments, the aptamer can be conjugated with fatty acids and / or coumarin derivatives. Conjugation of aptamer molecules with fatty acids and / or coumarin derivatives can significantly prolong their half-life in vivo.

[0077] In some embodiments, the fatty acid includes but is not limited to palmitic acid (PA), dodecanedioic acid (DA), tetradecanedioic acid, hexadecanedioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), etc. In some preferred embodiments, the fatty acid is octadecanedioic acid.

[0078] In some embodiments, the coumarin derivative includes but is not limited to 4-hydroxycoumarin, 3-acetyl-6-carboxycoumarin, warfarin, (2-oxo-2H-chromen-3-yl)acetic acid, [(8-acetyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetic acid, coumarin-3-carboxylic acid, N-(4-methyl-7-coumarin) oxalamide, 7-(carboxymethyl)-4-methylcoumarin, 7-methoxycoumarin-3-carboxylic acid, 6-methoxy-2-oxo-2H-chromen-3-carboxylic acid. In some preferred embodiments, the coumarin derivative is 4-hydroxycoumarin.

[0079] In some embodiments, the fatty acid, such as octadecanedioic acid, is conjugated to the 5' end of the aptamer. In some embodiments, the coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the 5' end of the aptamer. In some embodiments, the fatty acid, such as octadecanedioic acid, and the coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the 5' end of the aptamer.

[0080] In some embodiments, the fatty acid, such as octadecanedioic acid, is conjugated to the aptamer via a linker. In some embodiments, the coumarin derivative, such as 4-hydroxycoumarin, is conjugated to the aptamer via a linker. In some embodiments, the fatty acid, such as octadecanedioic acid, and the coumarin derivative, such as 4-hydroxycoumarin, are conjugated to the aptamer via a linker.

[0081] In some embodiments, the E3-specific ligand includes but is not limited to von Hippel-Landau (VHL), murine double minute 2 (MDM2), and cereblon (CRBN). In some preferred embodiments, the E3-specific ligand is VHL. For example, the VHL comprises the structure shown in the following formula:

[0082] In some embodiments, the E3-specific ligand is conjugated to the aptamer via a linker. In some embodiments, the linker is a bifunctional linker. The bifunctional linker can allow the aptamer to be conjugated to two different molecules, for example, a fatty acid and an E3-specific ligand.

[0083] In some embodiments, the bifunctional linker comprises one of the following structures:

[0084] In some embodiments, the linker comprises the following structure:

[0085] In some embodiments, the aptamer conjugate comprises the following structure: (FA / E3 ligand)-bifunctional linker-aptamer nucleotide sequence (5'-3'), wherein FA represents fatty acid, E3 ligand represents E3-specific ligand, and wherein FA and E3 ligand are conjugated to the 5' end of the aptamer nucleotide sequence via a bifunctional linker.

[0086] In some preferred embodiments, the fatty acid is octadecanedioic acid.

[0087] In some embodiments, the aptamer conjugate comprises a structure represented by the following formula:

[0088] In the structural formula of the present invention, the following structure represents the nucleotide sequence (5'-3' direction) of the aptamer (modified or unmodified):

[0089] In some embodiments, the aptamer conjugate comprises a structure represented by the following formula:

[0090] In some embodiments, the specific ligand of the asialoglycoprotein receptor (ASGPR) is N-acetylgalactosamine (GalNAc). In some embodiments, the ASGPR specific ligand, such as GalNAc, is conjugated to the aptamer via a linker.

[0091] In some embodiments, the aptamer conjugate comprises a structure represented by the following formula:

[0092] in

[0093] In some embodiments, the aptamer conjugate comprises a structure represented by the following formula:

[0094] In some preferred embodiments of various aspects of the present invention, the aptamer nucleotide sequence (5'-3' direction) is C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT, wherein (OMe) represents a 2'-methoxy (2'-OMe) modification of the corresponding nucleotide, and idT represents a 3' inverted deoxythymidine modification.

[0095] In some embodiments, the aptamer or aptamer conjugate of the present invention inhibits the biological activity of sclerostin. "Inhibit" means that the biological activity of sclerostin is reduced in the presence of the aptamer or aptamer conjugate compared to the absence of the aptamer or aptamer conjugate, for example, by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or even at least about 90%.

[0096] As used herein, the term "biological activity" refers to an effect on one or more cellular or extracellular processes that can affect physiological or pathophysiological processes. The biological activities of sclerostin include, but are not limited to, antagonizing the Wnt signaling pathway.

[0097] In some embodiments, the aptamer or aptamer conjugate of the present invention can inhibit the antagonistic effect of sclerostin on the Wnt signaling pathway. For example, the aptamer or aptamer conjugate of the present invention can block the antagonistic effect of sclerostin in a cell-based Wnt signaling assay.

[0098] In some embodiments, the aptamer or aptamer conjugate of the present invention inhibits the biological activity of sclerostin, such as the antagonism of sclerostin on the Wnt signaling pathway, with an EC50 value of less than 100 μg / ml, preferably less than 50 μg / ml, preferably less than 40 μg / ml, preferably less than 30 μg / ml, preferably less than 20 μg / ml, preferably less than 10 μg / ml or less. In some embodiments, the EC50 value is determined in vitro using a TOP-Wnt-inducible luciferase reporter gene assay in osteoblasts.

[0099] In some embodiments, the aptamer conjugates of the present invention are used for targeted degradation of sclerostin.

[0100] In some embodiments, the aptamer conjugate is used for targeted degradation of sclerostin in cells.

[0101] In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is a cancer cell that highly expresses sclerostin. In some embodiments, the cancer cell is a cancer cell that highly expresses sclerostin and asialoglycoprotein receptor (ASGPR). In some preferred embodiments, the cancer cell is a breast cancer cell (preferably a triple-negative breast cancer cell) or a liver cancer cell.

[0102] Disease treatment

[0103] In another aspect, the present invention provides a method for treating a disease by the aptamer conjugate of the present invention, the method comprising administering a therapeutically effective amount of the aptamer conjugate of the present invention to a subject in need thereof.

[0104] The disease treated by the aptamer conjugates of the present invention is, for example, a sclerostin-related disease, such as a sclerostin-mediated disease. In some embodiments, the disease is a disease caused by high expression of sclerostin.

[0105] As used herein, "sclerostin-associated diseases" include conditions in which bone mineral density (BMD) is abnormal and / or pathologically low relative to healthy subjects. Diseases characterized by low BMD and / or bone fragility include, but are not limited to, primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis (osteonecrosis), bone fractures and implant healing (dental implants and hip implants), bone loss due to other conditions (e.g., associated with HIV infection, cancer, and arthritis). Other "sclerostin-associated diseases" include, but are not limited to, hypophosphatemic rickets, rheumatoid arthritis, osteoarthritis, arthritis, and osteolytic lesions.

[0106] As used herein, "sclerostin-related diseases" include sclerostin-related cancers, such as cancers with high sclerostin expression. Examples of such cancers include, but are not limited to, myeloma (e.g., multiple myeloma with osteolytic lesions), breast cancer (e.g., triple-negative breast cancer), colon cancer, melanoma, liver cancer (hepatocellular carcinoma), epithelial cancer, esophageal cancer, brain cancer, lung cancer, prostate cancer, or pancreatic cancer, and any metastatic tumors thereof. In some embodiments, the cancer is breast cancer, preferably triple-negative breast cancer. In some embodiments, the cancer is liver cancer.

[0107] "Sclerostin-related diseases" may also include renal diseases and cardiovascular diseases caused by at least the expression of sclerostin in the kidney and in the cardiovascular system. Such conditions include, but are not limited to, renal diseases such as the following: glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, glomerular damage associated with systemic diseases such as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, polycystic kidney disease, neoplasia, sickle cell disease, and chronic inflammation), renal tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic kidney disease, medullary spongiosa renal, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug- and toxin-induced tubulointerstitial nephritis, hypercalcemic nephropathy, and hypokalemic nephropathy), acute and rapidly progressive renal failure, chronic renal failure, nephrolithiasis, gout, vascular diseases (e.g., hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic nephropathy, diffuse cortical necrosis, and renal infarction), or tumors (e.g., renal cell carcinoma and nephroblastoma).

[0108] The sclerostin-related diseases also include, but are not limited to, cardiovascular diseases such as ischemic heart disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, cor pulmonale, valvular heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., valve and vascular occlusive lesions, atrial or ventricular septal defects, and persistent ductus arteriosus), or cardiomyopathy (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy).

[0109] The subject can be any animal (domesticated, livestock or wild), including but not limited to cats, dogs, horses, pigs and cattle, and preferably a human subject. As used herein, the terms patient, individual and subject are used interchangeably.

[0110] The subject can be male or female. Preferably, the human subject is at risk for bone fracture, more preferably the human subject is at risk for or has osteoporosis. Preferably, the human subject is female, and more preferably female at risk for or has postmenopausal osteoporosis. It is expected that the methods of the present invention can be beneficial for subjects at any stage of osteoporosis.

[0111] As used herein, "treating" a subject having a disease means that the subject's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or the development of the disease.

[0112] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition. As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates, or improves the symptoms of a disease or condition, or cures the disease or condition.

[0113] The dosage regimen for using the aptamer conjugate is selected based on a variety of factors, including, for example, the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the specific aptamer conjugate or salt thereof used. An ordinarily skilled physician can readily determine and prescribe the effective amount of the composition required to prevent, counter, or inhibit the progression of the condition.

[0114] Typically, the dosage regimen of the aptamer conjugate is from about 1 μg / kg body weight to about 100 mg / kg body weight per day.

[0115] Exemplary treatment regimens require administration once a day, once every two days, once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three to six months, or a slightly shorter initial dosing interval (e.g., once a week to once every three weeks) followed by a longer dosing interval (e.g., once a month to once every three to six months). The frequency and interval of administration can be determined by those skilled in the art based on the pharmacokinetic parameters of the aptamer conjugate.

[0116] Pharmaceutical composition

[0117] In another aspect, the present invention also provides a pharmaceutical composition comprising at least one aptamer conjugate of the present invention and a pharmaceutically acceptable carrier or excipient, for example, for treating sclerostin-related diseases.

[0118] The aptamer conjugates described herein can be used in any pharmaceutically acceptable dosage form, including but not limited to injectable dosage forms, liquid dispersions, gels, sprays, ointments, creams, lyophilized formulations, dry powders, tablets, capsules, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, mixed immediate release and controlled release formulations, etc. Specifically, the aptamers described herein can be formulated to: (a) be administered by any one selected from oral, pulmonary, intravenous, intraarterial, intrathecal, intraarticular, rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal, intraperitoneal, topical, buccal, nasal, and local administration; (b) be in a dosage form selected from any one selected from liquid dispersions, gels, sprays, ointments, creams, tablets, sachets, and capsules; (c) be in a dosage form selected from any one selected from lyophilized formulations, dry powders, fast melt formulations, controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; or (d) any combination thereof.

[0119] Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may contain one or more of the following components: (1) a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; (2) an antibacterial agent, such as benzyl alcohol or methyl paraben; (3) an antioxidant, such as ascorbic acid or sodium sulfite; (4) a chelating agent, such as ethylenediaminetetraacetic acid; (5) a buffer, such as acetate, citrate, or phosphate; and (6) a substance for adjusting tonicity, such as sodium chloride or glucose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0120] The pharmaceutical composition that is suitable for injection use can comprise aseptic aqueous solution (wherein being water-soluble) or dispersion and the sterile powder for the temporary preparation of sterile injection solution or dispersion.For intravenous use, suitable carrier comprises physiological saline, antibacterial water or phosphate buffered saline (PBS).In all cases, described composition should be aseptic and its mobility should be easy to inject.Under the condition of manufacture and storage, described pharmaceutical composition should be stable and should be protected to prevent the contamination effect of microorganisms such as antibacterial and fungal.Term " stable " as used herein means to remain on the state or condition that is suitable for using to the patient.

[0121] In some embodiments, the carrier can be a solvent or dispersion medium, including water, ethanol, polyol (such as, glycerol, propylene glycol, liquid polyethylene glycol etc.) and a suitable mixture thereof. For example, by using a coating such as lecithin, by maintaining required particle size and by using a surfactant, suitable fluidity can be maintained. By various antibacterial and antifungal reagents, for example, p-hydroxybenzoate, chlorobutanol, phenol, ascorbic acid, thimerosal etc. can be realized to prevent the effect of microorganisms. In many cases, it is preferred to include isotonic agents in the composition, such as sugar, polyol (such as mannitol or sorbitol) and inorganic salts (such as sodium chloride). By including in the composition the material that delays absorption such as aluminum monostearate and gelatin, the absorption of the prolongation of the injectable composition can be brought.

[0122] Sterile injectable solutions can be prepared by incorporating the active agent (e.g., aptamer conjugate) in the desired amount with one or a combination of the ingredients listed above (as desired) in an appropriate solvent followed by filtration sterilization. Typically, dispersions are prepared by incorporating at least one aptamer conjugate into a sterile vehicle containing a basic dispersion medium and any other desired ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum drying and freeze drying, both of which yield a powder of the aptamer conjugate and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0123] Oral compositions typically include an inert diluent or edible carrier. For example, they can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the aptamers to sclerostin can be incorporated into excipients and used in the form of tablets, lozenges, or capsules. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition.

[0124] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., a gas (e.g., carbon dioxide), an atomized liquid, or a dry powder from a suitable device). For transmucosal or transdermal administration, a penetrant that is appropriate to the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active agent is formulated into an ointment, salves, gel, or cream as is known in the art. The agent can also be prepared in the form of a suppository (e.g., with a conventional suppository base, such as cocoa butter and other glycerides) or a retention enema for rectal delivery.

[0125] In one embodiment, the aptamer conjugate is formulated for topical administration. As used herein, "topical administration" refers to delivering the aptamer conjugate to the animal by contacting (directly or otherwise) a formulation comprising the aptamer conjugate with all or part of the skin (epidermis) of the animal. The term encompasses several routes of administration, including, but not limited to, topical administration and transdermal administration. A common requirement for these modes of administration is effective delivery to the target tissue or layer. On the one hand, topical administration is used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the aptamer conjugate. On the other hand, topical administration is used as a means to selectively deliver the aptamer conjugate to the epidermis or dermis of an animal or a specific layer thereof.

[0126] For topical administration, the aptamer conjugates can be formulated into pharmaceutically acceptable ointments, creams, lotions, eye ointments, eye drops, ear drops, impregnated dressings, and aerosols, medicated powders, medicated adhesives, foams, and can contain appropriate conventional additives or excipients, including, for example, preservatives or solvents to aid drug penetration and emollients in ointments, gels, and creams. Such topical formulations may also contain compatible conventional carriers, such as ethanol or oleyl alcohol for emulsions. Such carriers may constitute from about 1% to about 98% by weight of the formulation, and more typically, such carriers will constitute up to about 80% by weight of the formulation. Specific formulations for topical delivery of aptamers are described in the prior art.

[0127] In one embodiment, the aptamer conjugate is prepared with a carrier that prevents rapid removal from the body. For example, a controlled release formulation can be used, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0128] Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0129] In addition, suspensions of the aptamer conjugates can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate, triglycerides) or liposomes. Non-lipid polycationic amino acid polymers can also be used for delivery. Optionally, the suspension can also include a suitable stabilizer or agent to increase the solubility of the compound and allow for the preparation of highly concentrated solutions.

[0130] In some cases, it may be particularly advantageous to formulate oral or parenteral compositions in dosage units for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of aptamer conjugate calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification of dosage unit forms for the aptamer conjugates described herein is dictated by and directly dependent upon the unique characteristics of the particular aptamer conjugate and the particular therapeutic effect to be achieved, as well as the inherent limitations of the art of formulating such active agents for use in treating individuals.

[0131] Pharmaceutical compositions comprising at least one aptamer conjugate may include one or more pharmaceutical excipients. Examples of such excipients include, but are not limited to, binders, fillers, lubricants, suspending agents, sweeteners, flavorings, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Exemplary excipients include: (1) binders, including various celluloses and cross-linked polyvinyl pyrrolidone, microcrystalline cellulose (such as Avicel PH101 and Avicel PH102), silicified microcrystalline cellulose (ProSolv SMCC TM), tragacanth and gelatin; (2) fillers, such as various starches, lactose, lactose monohydrate, anhydrous lactose; (3) disintegrants, such as alginic acid, Primogel, corn starch, lightly cross-linked polyvinyl pyrrolidine, potato starch, corn starch and modified starches, cross-linked sodium carboxymethylcellulose, crospovidone, sodium starch glycolate and mixtures thereof; (4) lubricants, including agents that affect the flowability of the powder to be compressed, including magnesium stearate, colloidal silicon dioxide (such as Aerosil 200, talc), stearic acid, calcium stearate and silica gel; (5) glidants, such as colloidal silicon dioxide; (6) preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid (such as butylparaben), alcohols (such as ethanol or benzyl alcohol), phenolic compounds (such as phenol) or quaternary ammonium compounds (such as benzalkonium chloride); (7) diluents, such as pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, calcium hydrogen phosphate, sugars and / or any mixture thereof; examples of diluents include microcrystalline cellulose, such as Avicel PH101 and Avicel PH102; lactose, such as lactose monohydrate, anhydrous lactose and Pharmatose DCL21; calcium hydrogen phosphate such as Emcompress mannitol, starch, sorbitol, sucrose and glucose; (8) sweeteners, including any natural or artificial sweeteners, such as sucrose, saccharin sucrose, xylitol, saccharin sodium, sodium cyclamate, aspartame and acesulfame potassium; (9) flavorings, such as mint, methyl salicylate, orange flavoring, Magnasweet (trademark MAFCO), bubble gum flavor, fruit flavoring, etc.; and (10) effervescent agents, including effervescent agent pairs, such as organic acids and carbonates or bicarbonates. Example

[0132] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples.

[0133] Example 1: E3 ligand VHL linked to OA-conjugated sclerostin aptamer can significantly degrade intracellular sclerostin in MDA-MB-231 cells in vitro

[0134] The present inventors identified a DNA-sclerostin aptamer (termed Apc001) that promotes bone formation in vivo (Wang et al., 2022; Yu et al., 2022). Subsequently, a long-acting Apc001 (Apc001OA) was developed by modification with octadecanedioic acid (PCT / CN2022 / 082996) (Zhang et al., 2022b). SOST mRNA expression has been reported in most clinical TNBC (triple-negative breast cancer) tissues, but not in healthy breast tissue (Hesse et al., 2019). Recently, the inventors' in vitro data demonstrated that octadecanedioic acid promoted the internalization of conjugated Apc001 in MDA-MB-231 cells (TNBC cells) in vitro. Surprisingly, Apc001OA inhibited MDA-MB-231 cell proliferation in vitro, whereas sclerostin antibodies did not. This suggests that the role of intracellular sclerostin in the in vitro proliferation of MDA-MB-231 cells has remained largely unrecognized. The present inventors hope to address the scientific question of whether intracellular sclerostin can become a promising therapeutic target for treating TNBC. It is necessary to develop a tool for in vitro targeted degradation of intracellular sclerostin for TNBC.

[0135] Proteolysis-targeting chimera (PROTAC) technology is a novel therapeutic strategy that has attracted widespread attention from academia and the pharmaceutical industry (Garber, 2022). PROTAC degraders consist of two ligands linked by a linker: one ligand binds to the protein of interest (POI) and the other binds to an E3 ligase. By forming a ternary complex of POI-PROTAC-E3 ligase, PROTACs hijack the ubiquitin-proteasome system (UPS) to degrade POIs (Sakamoto et al., 2001).

[0136] Therefore, a sclerostin aptamer-linked E3 ligand was designed to degrade intracellular sclerostin in MDA-MB-231 cells in vitro. Apc001OA is an ideal sclerostin ligand because it has high binding affinity to sclerostin and is internalized in MDA-MB-231 cells in vitro. The VHL ligand is a widely used E3 ligand in PROTACs (Cao et al., 2022), which was linked to Apc001OA using a PEG linker with good solubility.

[0137] The purpose of this example is to compare the differences in intracellular sclerostin expression levels in MDA-MB-231 cells treated with Apc001OA-VHL and PBS by Western blot analysis.

[0138] Experimental design

[0139] Objective Experimental Design: To chemically obtain a VHL ligand linked to a sclerostin aptamer, the 5'-alkyne Apc001OA aptamer was conjugated to azide-linked E3 ligands (including VHL ligands) via a copper click reaction. Following completion of the reaction, the reaction mixture was purified on a Sephadex™ G-25 DNA-grade column and HPLC. The VHL E3 ligand linked to Apc001OA (Apc001OA-VHL) was obtained and confirmed by electrospray ionization (ESI-MS).

[0140] MDA-MB-231 cells were plated at 4×10 5 Cells were seeded at a density of 100 μM in 6-well culture dishes and incubated overnight. The cells were then incubated with either 0.7 μM Apc001OA-VHL or 1.4 μM Apc001OA-VHL, respectively, and PBS at 37°C. After 24 hours, the supernatant was removed and protein extracts were isolated from the cells. Finally, protein samples were analyzed by Western blotting.

[0141] Evaluation Plan

[0142] Synthesis of the designed VHL E3 ligand linked to OA-conjugated sclerostin aptamer: Apc001OA (1.0 eq), azide-linked E3 ligand (100 eq), and CuSO4 (50 eq) were transferred to a 1.5 mL plastic reaction tube. NaHCO3 (5 μL, 200 mM) and CH3CN / H2O (100 μL, VHL E3 ligand) were then added. CH3CN / V H2O =6%), and the mixture was vortexed briefly. Next, TCEP (3 μL, 20 mM) was added and the mixture was further vortexed. The reaction tube was then briefly purged with nitrogen and kept on a shaker at 25 ° C for 4 hours (Patil et al., 2021). After the reaction was completed, the reaction mixture was purified by SephadexTM G-25 DNA Grade Columns. The crude product was then further purified on HPLC by a gradient method using 0.5 M TEAA and acetonitrile as solvents. Appropriate fractions were collected and confirmed by ESI-MS analysis.

[0143] Western Blot Analysis: All protein samples were separated by denaturing 10% SDS-PAGE gels at 80 V for 30 minutes, followed by 120 V for 1 hour. Gels were transferred to 0.2 μm PVDF membranes (Bio-rad) by electroblotting at 350 A for 60 minutes. All membranes were blocked with 5% blotting-grade blocking reagent (Bio-rad) containing 0.1% Tween 20 (TBST) at room temperature for 1 hour and then incubated with primary antibodies overnight at 4°C. Membranes were incubated with peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Bound antibodies were visualized by enhanced chemiluminescence (ECL) system (Bio-rad) according to the manufacturer's instructions. Sclerostin was detected by anti-sclerostin polyclonal antibody (Abcam, #ab85799). GAPDH was used as a control and was detected by anti-GAPDH monoclonal antibody (Santa Cruze, #sc365062). All Western blot images were processed using Image Lab software.

[0144] Results and Discussion

[0145] Synthesis of the Designed VHL E3 Ligand Linked to the OA-Conjugated Sclerostin Aptamer: 5'-alkyne Apc001OA was linked to an azide-linked E3 ligand via a copper click reaction to synthesize the VHL E3 ligand linked to the sclerostin aptamer (Figure 1A). After the copper click reaction, the crude product was purified on a Sephadex™ G-25 DNA-grade column and HPLC (Figure 1B), and the purified product was confirmed by ESI-MS (Figure 1C). These data demonstrate that the VHL E3 ligand linked to the sclerostin aptamer was successfully synthesized.

[0146] Western blot analysis: Western blot analysis showed that in MDA-MB-231 cells, the expression of intracellular sclerostin in the 0.7 and 1.4 μM Apc001OA-VHL treatment groups was lower than that in the PBS-treated group (Figure 2). This indicates that the E3 ligand VHL linked to the OA-conjugated sclerostin aptamer can significantly degrade intracellular sclerostin in MDA-MB-231 cells in vitro.

[0147] Example 2: The degradation of intracellular sclerostin by the E3 ligand VHL linked to the OA-conjugated sclerostin aptamer persists in MDA-MB-231 cells in vitro

[0148] The purpose of this example is to compare the differences in the expression levels of sclerostin in MDA-MB-231 cells after treatment with 0.7 μM Apc001OA-VHL for 6 h, 12 h, 24 h, and 48 h, respectively, by Western blot analysis.

[0149] Experimental design

[0150] MDA-MB-231 cells were plated at 4×10 5 Cells were seeded at a density of 100 μM in 6-well culture dishes and incubated overnight. Cells were incubated with 0.7 μM Apc001OA-VHL and harvested at 37°C for 6, 12, 24, and 48 hours. The supernatant was then removed and protein extracts were isolated from the cells. Protein samples were analyzed by Western blotting.

[0151] Evaluation Plan

[0152] Western Blot Analysis: All protein samples were separated by denaturing 10% SDS-PAGE gels at 80 V for 30 minutes, followed by 120 V for 1 hour. Gels were transferred to 0.2 μm PVDF membranes (Bio-rad) by electroblotting at 350 A for 60 minutes. All membranes were blocked with 5% blotting-grade blocking reagent (Bio-rad) containing 0.1% Tween 20 (TBST) at room temperature for 1 hour and then incubated with primary antibodies overnight at 4°C. Membranes were incubated with peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Bound antibodies were visualized by enhanced chemiluminescence (ECL) (Bio-rad) according to the manufacturer's instructions. Sclerostin was detected by anti-sclerostin polyclonal antibody (Abcam, #ab85799). GAPDH was used as a control and was detected by anti-GAPDH monoclonal antibody (Santa Cruze, #sc365062). All Western blot images were processed using Image Lab software.

[0153] Results and Discussion

[0154] Western blot analysis showed that the intracellular sclerostin level in the Apc001OA-VHL-treated group was significantly lower than that in the PBS-treated group within 24 hours (Figure 3). Western blot analysis showed that the lowest intracellular sclerostin level was observed 6 hours after 0.7 μM Apc001OA-VHL treatment. After VHL treatment, the intracellular sclerostin level gradually recovered from 12 to 48 hours (Figure 3). This may be explained by the following two reasons. One of them may be related to the de novo synthesis of sclerostin in MDA-MB-231 cells. The other may be related to the instability of Apc001OA-VHL in MDA-MB-231 cells. These data indicate that the degradation effect of the designed E3 ligand VHL linked to the OA-conjugated sclerostin aptamer on intracellular sclerostin can be sustained for 24 hours in MDA-MB-231 cells in vitro.

[0155] Example 3: E3 ligand VHL linked to OA-conjugated sclerostin aptamer inhibits proliferation and migration of MDA-MB-231 cells in vitro

[0156] Objective 1 of this example: to compare the differences in cell proliferation of MDA-MB-231 cells treated with Apc001OA, Apc001OA-VHL, and PBS, respectively, through in vitro colony formation.

[0157] Objective 2 of this example: To compare the differences in cell migration of MDA-MB-231 cells treated with Apc001OA, Apc001OA-VHL, and PBS, respectively, by using an in vitro Transwell assay.

[0158] Experimental design

[0159] Experimental design for objective 1:

[0160] MDA-MB-231 cells were seeded in 6-well culture dishes and incubated overnight. Cells were incubated with Apc001OA-VHL (700 nM), Apc001OA (700 nM), and PBS at 37°C. The culture medium was changed every 2 days. Colonies were analyzed after 9 days.

[0161] Experimental design for Aim 2:

[0162] MDA-MB-231 cells were suspended in serum-free DMEM medium and then distributed into the upper chamber of a 24-well transwell plate. 700 μl of DMEM medium supplemented with 10% FBS was added to each well of the lower 24-well plate. The cells were incubated with Apc001OA-VHL (700 nM), Apc001OA (700 nM), and PBS at 37°C for 48 hours. At the end of the incubation period, the number of migrated cells in each transwell chamber was analyzed.

[0163] Evaluation Plan

[0164] Colony formation assay: MDA-MB-231 cells were trypsinized and counted, and 1000 cells were seeded into 6-well plates containing complete DMEM medium. Cells were then treated with PBS, Apc001OA-VHL, and Apc001OA, respectively, with the medium changed every 2 days until visible colonies formed. After 9 days, cells were fixed with 4% PFA for 10 minutes and stained with crystal violet for 20 minutes (Beyotime). Images were captured using a camera.

[0165] Migration assay: MDA-MB-231 cells were suspended in serum-free DMEM medium and counted, and then 5x10 5Cells were distributed into the upper chamber of a 24-well transwell. 700 μL of DMEM supplemented with 10% FBS was added to each well of the lower 24-well plate. The cells were incubated with Apc001OA-VHL, Apc001OA, and PBS at 37°C for 48 hours. At the end of the incubation period, the cells were fixed with 4% PFA for 10 minutes and stained with crystal violet for 20 minutes (Beyotime). The number of migrated cells in each transwell chamber was analyzed microscopically.

[0166] Results and Discussion

[0167] Colony formation assays showed that the 700 nM Apc001OA-VHL treatment group exhibited fewer colonies than the PBS and 700 nM Apc001OA treatment groups (Figure 4). Migration assays showed that the 700 nM Apc001OA-VHL treatment group exhibited fewer migrating cells than the PBS and 700 nM Apc001OA treatment groups (Figure 5). These data demonstrate that the E3 ligand VHL linked to the OA-conjugated sclerostin aptamer (Apc001OA-VHL) can inhibit cell proliferation and migration of MDA-MB-231 cells in vitro.

[0168] Example 4: E3 ligand VHL linked to OA-conjugated sclerostin aptamer inhibits cell viability of MDA-MB-231 cells in vitro

[0169] The purpose of this example is to compare the differences in cell viability of MDA-MB-231 cells treated with Apc001OA, Apc001OA-VHL and PBS respectively by WST-8 assay.

[0170] Evaluation Plan

[0171] WST-8 assay: Cell viability was assessed using the lactate dehydrogenase-based WST-8 assay (Dojindo Molecular Technologies) (Qin et al., 2018) using a Tecan Infinite M1000 multimode microplate reader (Tecan, Morrisville, NC). The WST-8 reagent was added to 96-well culture plates, incubated at 37°C for 1 hour, and read at 450 nm. Readings were normalized to PBS-treated cells.

[0172] Results and Discussion

[0173] WST-8 analysis showed that the cell viability of the 700 nM Apc001OA-VHL treatment group was lower than that of the PBS and 700 nM Apc001OA treatment groups (Figure 6). These data indicate that the designed E3 ligand VHL linked to the OA-conjugated sclerostin aptamer (Apc001OA-VHL) can inhibit the cell viability of MDA-MB-231 cells in vitro.

[0174] Example 5: E3 ligand VHL linked to OA-conjugated sclerostin aptamer inhibits tumor growth in a subcutaneous mouse model inoculated with MDA-MB-231 cells and tumor metastasis in an orthotopic mouse model of 4T1 cells

[0175] Evaluation Plan

[0176] MDA-MB-231 subcutaneous mouse model. For mammary fat pad injection experiments, all female athymic nude mice aged 6-8 weeks were anesthetized under 2.5% isoflurane and then injected with MDA-MB-231 (2×10 6 ) were injected subcutaneously (sc) and mice were tested using a single cell suspension in 50 μl of Matrigel:PBS (1:1). Tumor size was monitored by measuring tumor length (L) and width (W) with a caliper and calculating tumor volume using the formula: V = L x W 2 / 2. Construct tumor growth curve to evaluate primary tumor progression. When the total tumor volume exceeds 1000mm 3 All experiments on individual mice were terminated at 4 hr, or earlier if the tumor ulcerated. At the end of the experiment, the primary tumor was excised, photographed, and weighed.

[0177] 4T1 orthotopic mouse model. All 8-week-old female Balb / c mice were anesthetized under 2.5% isoflurane, and 4T1-luc sost WT cells (1× 10 5 ) was injected subcutaneously (sc) into the abdominal mammary gland. After all experiments on individual mice were completed, lung tissues were collected and lung nodules were counted.

[0178] Results and Discussion

[0179] To investigate the effect of Apc001OA-VHL on TNBC progression in vivo, a mouse model inoculated with MDA-MB-231 cells was treated with PBS, Apc001OA, and Apc001OA-VHL, respectively. The treatment regimen for mice inoculated with MDA-MB-231 cells was described. Tumor volume measurements (Figure 7) showed that the tumor volume in the Apc001OA-VHL group was significantly smaller than that in the PBS (p<0.001) and Apc001OA (p<0.01) groups. Tumor weight measurements (Figure 7) showed that the tumor weight in the Apc001OA-VHL group was significantly lighter than that in the PBS (p<0.01) and Apc001OA (p<0.05) groups. These data indicate that Apc001OA-VHL's degradation of intracellular sclerostin significantly inhibited tumor progression in the mouse model inoculated with MDA-MB-231 cells.

[0180] To investigate the effect of Apc001OA-VHL on TNBC metastasis in vivo, mice inoculated with 4T1 cells were treated with PBS, Apc001OA, and Apc001OA-VHL. Visible lung nodules were counted to quantify lung metastasis. Statistical results showed that the number of visible lung metastatic nodules was significantly reduced in the Apc001OA-VHL group compared with the PBS and Apc001OA groups (Figure 7). This suggests that Apc001OA-VHL's degradation of intracellular sclerostin significantly inhibits tumor metastasis in the orthotopic mouse model inoculated with 4T1 cells.

[0181] Example 6. Synthesis and Application of Nucleic Acid-Based Molecules (LYTACs) for Targeted Extracellular and Systemic Protein Degradation

[0182] At present, targeted protein degradation (TPD) has become a very promising targeted therapy technology, which is mainly based on two protein degradation pathways: the proteasome pathway and the lysosome pathway. For the proteasome pathway, the bifunctional molecule that connects the target protein and the E3 enzyme is called a protein hydrolysis targeting chimera (PROTAC), which uses the ubiquitin-proteasome system to degrade the intracellular target protein through a series of enzymes. For the lysosomal pathway, the bifunctional molecule that connects the target protein and the transmembrane receptor is the most well-known lysosomal targeting chimera (LYTAC), which mediates the internalization of the target protein into the lysosome after endocytosis. The lysosomal microenvironment then degrades the target transmembrane or extracellular protein, which is a good supplement to PROTAC.

[0183] Inspired by the earliest PROTAC molecules, other TPD technologies, such as autophagy-targeting chimeras (AUTACs), specific and nongenetic apoptosis inhibitor protein-dependent protein erasers (SNIPERs), and autophagosome-tethering compounds (ATTECs), are emerging and continue to develop. Among them, the trivalent N-acetylgalactosamine antibody conjugate (GalNAc-Antibody) LYTAC developed by Bertozzi's group has attracted widespread attention in the academic community for its targeted protein degradation by binding to the asialoglycoprotein receptor (ASGPR). ASGPR is highly expressed on the surface of hepatocytes and can recognize the GalNAc moiety and internalize its conjugate into lysosomes via clathrin-mediated endocytosis, making it a well-characterized liver-specific lysosomal targeting receptor. It has been widely used for liver-targeted oligonucleotide delivery via GalNAc recognition. In their work, the Bertozzi group successfully degraded epidermal growth factor receptor (EGFR) in vitro with GalNAc-Antibody LYTAC, and integrin in vitro with GalNAc-pipetide LYTAC. However, the conjugation of antibodies to GalNAc molecules to prepare chimeras is not only complex and time-consuming, with uncertain conjugation number and position, but also has a large molecular weight (usually around 150 kD), resulting in poor cellular internalization.

[0184] Aptamers are short fragments of DNA or RNA that can recognize and bind to target molecules (usually proteins) through their specific 3D structures. These fragments are selected from randomly synthesized oligodeoxynucleotides or oligonucleotide libraries using SELEX (systematic evolution of ligands by exponential enrichment). Compared to small molecule binders, aptamers often have better selectivity and higher affinity. Compared to antibodies, aptamers have a simpler evolution process, are easy to modify, have affinity adjustments, have low immunogenicity, and are versatile in structural design and engineering, making them ideal recognition ligands for their targets. As a result, an increasing number of aptamers are being developed and used as therapeutic agents and probes.

[0185] Following the development of GalNAc-Antibody LYTAC by Bertozzi's group, Zhu Zhi's group developed GalNAc-Aptamer, which achieves hepatocyte-specific degradation of membrane protein tyrosine kinase 7 (PTK7) and extracellular protein platelet-derived growth factor (PDGF) by conjugating GalNAc molecules with corresponding aptamers. However, they only discussed the feasibility of GalNAc-Aptamer for TPD in vitro. Specific in vivo application scenarios are urgently needed to verify the drugability of GalNAc-Aptamer-mediated degradation of pathogenic proteins.

[0186] Evaluation Plan

[0187] Construction of GalNAc-Apc001 conjugate (Figure 8): N-hydroxysuccinimide (NHS) (50.7 mg, 0.45 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (85.7 mg, 0.45 mmol) were added to a stirred solution of tris-GalNAc carboxylic acid derivative (Compound 1, WuXi AppTec) (500 mg, 0.3 mmol) in anhydrous dichloromethane (DCM) (20 mL) under nitrogen protection. The mixture was allowed to stand overnight and then quenched by adding saturated NaHCO 3. The residue was quickly partitioned between DCM (100 mL) and saturated NaHCO 3 (50 mL). The organic layer was washed with saturated NaCl, dried (Na 2 SO 4) and evaporated to dryness to give the crude product (Compound 2) (540 mg, 0.246 mmol, yield ~82%). To a solution of amino-DNA (100 nmol) in triethylamine acetate (TEAA) (1.0 M, 5 mL, pH = 8.0), compound 2 was added (without further manipulation) and allowed to stand overnight. The mixture was then added to an ammonium hydroxide solution (10 mL) and heated to 60° C. within 3 hours. The solvent was removed in vacuo, and the solid residue was redissolved in dH2O (1 mL). The mixture was then purified using high performance liquid chromatography (Agilent 1260). Phase A was acetonitrile (CAN) and phase B was TEAA (50 mM). A gradient of 5% to 60% phase A was run over a BEH C18 OBD™ preparative oligonucleotide column (2.5 μm, 10 mm × 50 mm) over 30 minutes at a flow rate of 1.2 mL min⁻¹ at ambient temperature. Purified GalNAc-Apc001 (or GalNAc-scApc001, negative control) was desalted using a Sephadex G25 column to yield compound 3. The sample was then lyophilized for storage.

[0188] Confocal laser scanning microscopy (CLSM) imaging: HepG2 and A373 cells were imaged at 8×10 4After the density of 100 μg / ml was seeded on the confocal plate in 2mL DMEM culture medium, it was cultured for 24 hours at 37°C, humidified air and 5% CO2. Then the preset concentration of GalNAc-Apc001-FAM, Apc001-FAM, GalNAc-Scramble, GalNAc-Apc001 / Sclerostin-FAM mixture, Apc001 / Sclerostin-FAM mixture or GalNAc-Scramble / Sclerostin-FAM mixture was added to the culture medium, and the cells were incubated for a preset time. Afterwards, the nucleus was stained with 1×Hoechst 33342 staining solution (Biyotime) and the lysosome was stained with 50nM Lysotracker Red (Biyotime) at 37°C for 30 minutes. The cells were gently washed 3 times with 1×PBS buffer and the cells were retained in 1×PBS buffer throughout the imaging process. Fluorescence imaging of cells was performed using a confocal laser scanning microscope (LEICA TCS SP8) in water immersion mode at 60x magnification using selected channels (blue channel: 405 nm; green channel: 488 nm; red channel: 640 nm). Note: For time-dependent imaging, nuclear and lysosomal staining was not performed to enhance cell viability. At each imaging time point, the medium containing the fluorescent sample was replaced with normal medium and reversed for further incubation after imaging was completed.

[0189] Western blot analysis of the accumulation and degradation of sclerostin in ASGPR+ cells: HepG2 cells were cultured at 1×10 5After being seeded on a confocal plate in a 24-well plate at a density of 1 / well, the cells were cultured at 37°C in a humidified atmosphere of 5% CO2 for 24 hours. The cells were then incubated with a 500nM GalNAc-Apc001 / Sclerostin (1:1) mixture for different times (0 hour, 1 hour, 2 hours, 4 hours, 8 hours) as needed. At the 8-hour time point, the culture medium was replaced with normal culture medium to observe the degradation of sclerostin until 18 hours. The cells were washed three times with 1× PBS buffer and lysed with RIPA buffer on ice for 30 minutes. The supernatant was collected and the protein concentration was roughly calculated using a Nanodrop 2000. Equal amounts of cell lysates were electrophoresed through a 12% SDS-PAGE gel, then transferred to a PVDF membrane and nonspecifically blocked with 5% skim milk in TBST buffer. The membrane was then incubated with the primary antibody (1:1000 diluted in 5% skim milk) at 4°C overnight. The membrane was gently washed three times with 1× TBST buffer on a shaker and incubated with secondary antibody (1:5000 diluted in 5% skim milk) at room temperature for 1 h. After washing three times with TBST buffer, the membrane was treated with BeyoECL Plus substrate for 2 minutes and imaged using ChemiDoc Imaging Systems (Bio-Rad).

[0190] Evaluation of the effect of GalNAc-Apc001 on sclerostin-induced Wnt signaling inhibition: HEK293 cells were cultured at 37°C in a humidified atmosphere with 5% CO2 using Dulbecco's modified Eagle's medium (DMEM, Omacgene) containing 10% fetal bovine serum (FBS, CellMax) and 1% penicillin-streptomycin (Gibco by Life Technology). 5The density of 1:1 is inoculated in the 24-well plates in 0.5mL DMEM culture medium, cultivated 24 hours.Use lipofectamine 3000 that Top flash plasmid (Firefly luciferase, 100ng / hole), SV40 plasmid (Renilla luciferase, 10ng / hole) and Wnt-1 plasmid (200ng / hole) cotransfection is in cell (Lipofectamine 3000 1 μ L / hole, P3000 reagent 1 μ L / hole, Thermoscientific).In this process, first plasmid and lipofectamine 3000 are mixed as buffer A in 25 μ L OptiMem culture medium (Omacgene).P3000 reagent is dissolved in 25 μ L OptiMem culture medium as buffer B.Buffer A and buffer B are mixed and hatched 15 minutes.Then mixture is directly added in culture medium and cultivated 6 hours. Subsequently, the transfection medium was removed and 0.5 mL of fresh DMEM medium containing 100 nM sclerostin and 2 × 10 5 Other cells (HepG2, A375, or none) and 500 nM samples (GalNAc-Apc001, Apc001, GalNAc-Scramble, or Sclerosrin Antibody) that require validation were added. After incubation at 37°C for 12 hours, the medium was removed, and the culture vessel was shaken at room temperature for 15 minutes. The cells were lysed in 200 μL of 1× passive lysis buffer (PLB, Promega). Then, 15 μL of the lysate was transferred to a 96-well OptiPlate, and luciferase activity was measured by measuring chemiluminescence using the MD SpectraMax i3X Multi-Mode Microplate Reader System according to the manufacturer's protocol of the Dual-Luciferase Reporter Gene Assay System (Promega).

[0191] MicroCT analysis: Bone mass and trabecular microarchitecture were analyzed using microCT (version 6.5, vivaCT40, SCANCO Medical AG, Bassersdorf, Switzerland) for the cortical microarchitecture of the left femoral midshaft, the trabecular bone of the left proximal tibial metaphysis, and the trabecular bone of the left distal femoral metaphysis. Images of the femur and tibia were reconstructed and calibrated with an isotropic voxel size of 12.5 and 17.5 μm, respectively (70 kVp, 114 μA, 200 ms integration time, 260 threshold, 1200 mg HA / cm3). The same filtering and segmentation values ​​were used for each measurement. Using Scanco evaluation software, regions of interest (ROIs) were defined for trabecular parameters. For trabecular bone, a central region equivalent to 70% of the vertebral body height was selected and extended from the proximal to the distal end of the growth plate toward the vertebral body. For the proximal tibia and distal femur of mice, 100 consecutive sections starting from 0.1 mm proximal to the growth plate, where the two condyles were no longer visible, were selected for analysis. Trabecular ROIs were drawn freehand on 100 consecutive sections to ensure that they were within the endosteal envelope. Trabecular bone parameters, including trabecular volumetric bone mineral density (Tb.vBMD), trabecular number (Tb.N), and trabecular connection density (Tb.conn.D), were calculated. For the femoral midshaft, 100 consecutive sections were measured at the exact center and distal 50% of the femoral length using an automatic threshold algorithm. Trabeculae in contact with cortical bone were manually removed from the ROI. Cortical bone parameters, including Ct. periosteal circumference, Ct. endosteal circumference, and Ct. bone strength index, were calculated.

[0192] Statistical Analysis: All variables are expressed as mean ± standard deviation. One-way analysis of variance with Tukey's post hoc test was performed to identify between-group differences in study variables, including in vitro Wnt-induced signaling, in vitro bone formation biomarker mRNA levels, Micro-CT parameters, bone histomorphometric parameters, and mechanical testing. All statistical data were analyzed using Origin 2019b and GraphPad Prism, with P < 0.05 considered statistically significant. For in vivo experiments, animals were randomly assigned to groups. Animals in poor physical condition were excluded.

[0193] Results and Discussion

[0194] Transmembrane transport potential of GalNAc-Apc001 conjugate in ASGPR+ cells

[0195] Tri-GalNAc has been widely used to deliver a variety of cargoes to hepatocytes through cellular uptake mediated by the asialoglycoprotein receptor (ASGPR). In order to determine the transmembrane transport potential of the GalNAc-Apc001 conjugate, GalNAc-Apc001 was fluorescently labeled with a FAM group at the 3' end, i.e., GalNAc-Apc001-FAM. Apc001-FAM and the scrambled sequence GalNAc-Scramble-FAM were used as controls. HepG2 cells, a liver cancer cell line reported to have high levels of ASGPR expression on the cell surface, were used as positive cells. In contrast, A375 cells, a human melanoma cell line with the lowest expression of ASGPR, were used as negative cells. Confocal laser scanning microscopy imaging was performed to determine the transmembrane transport potential of the GalNAc-Apc001 conjugate in ASGPR+ cells. After HepG2 cells were incubated with GalNAc-Apc001-FAM for 2 hours, an increase in the fluorescence signal was clearly seen, while no increase in the fluorescence signal was observed in A375 cells (Figure 9), indicating that the internalization of GalNAc-Apc001 was cell-specific. Both GalNAc-Apc001-FAM and GalNAc-Scramble-FAM treatment increased the fluorescence signal in HepG2 cells, while no increase in the fluorescence signal was observed in the Apc001-FAM-treated group, indicating that internalization was GalNAc-dependent (Figure 9). In addition, the internalization fluorescence signal in GalNAc-treated HepG2 cells co-localized with the Lysotracker Red signal used to visualize acidic lysosomes, indicating that GalNAc mediates its conjugated cargo to the acidic compartments in ASGPR+ cells. In summary, GalNAc can mediate cell-specific internalization and is able to transport FAM-labeled Apc001 to ASGPR+ cell lysosomes.

[0196] GalNAc-Apc001 conjugate targets sclerostin for degradation in ASGPR+ cells

[0197] Western blotting of sclerostin (GAPDH as an internal control) was performed to monitor changes in the content of HepG2 cells. As shown in Figure 10, time-dependent accumulation of sclerostin in HepG2 cells was observed under culture sample medium (1 hour to 8 hours). After the sample medium was replaced with normal medium at the 8-hour time point, gradual degradation of sclerostin was observed (8 hours to 18 hours). This indicates that GalNAc-Apc001 can mediate cell-specific degradation of extracellular sclerostin in ASGPR+ cells.

[0198] In the presence of ASGPR+ cells in vitro, GalNAc-Apc001 can greatly attenuate the inhibitory effect of sclerostin on Wnt signaling.

[0199] The HEK293 cell line has been widely used as a cell tool cell line for Wnt signaling research and was incubated for 6 hours with Top flash plasmid (Firefly luciferase, TCF / LEF response element), SV40 plasmid (Renilla luciferase, internal control) and Wnt-1 plasmid. After replacing the culture medium, sclerostin was added directly to the culture medium to construct a model in which Wnt signaling induced by Wnt-1 was greatly inhibited by sclerostin. After treating the cells with sclerostin inhibitors, Wnt signaling was reactivated. As expected, the data showed that Wnt-1-induced luciferase signaling was greatly inhibited by sclerostin, confirming the inhibitory effect of sclerostin on Wnt signaling. To evaluate the inhibition of Wnt signaling induced by sclerostin by GalNAc-Apc001, other cells (HepG2, A375, or none) and the sample mixture to be verified (GalNAc-Apc001, Apc001, GalNAc-Scramble, or sclerostin antibody) prepared in advance were added to the culture medium after transfection. After incubation for 12 hours, the cells were lysed and luciferase activity was measured by a dual reporter gene assay. Surprisingly, in the presence of HepG2 cells, the relative luciferase activity of the GalNAc-Apc001-treated group was restored to 96% of Wnt-1 induction, nearly 15% higher than the group treated with the sclerostin antibody Romosozumab, and 39% higher than the group treated with Apc001. However, no significance was observed between GalNAc-Apc001 and Apc001 in the presence of A375 cells or in the absence of additional cells. This suggests that GalNAc-Apc001 induces Wnt signaling reactivation specifically in ASPGPR+ cells. Furthermore, no Wnt signaling reactivation was observed in the GalNAc-Scramble treatment group, regardless of the presence of HepG cells, A375 cells, or other cells, indicating that Wnt signaling reactivation is Apc001-dependent.

[0200] GalNAc conjugation facilitates the modified aptamer to promote bone anabolism in osteogenesis imperfecta mice (Col1a2 + / G610C )

[0201] To evaluate the effect of GalNAc-modified aptamers on promoting bone anabolism in OI mice, micro-computed tomography (micro-CT) analysis was used to measure Col1a2 + / G610CTrabecular bone of the distal femoral metaphysis of mice. Six to eight-week-old OI mice were subcutaneously injected (once a week for 12 weeks) with PBS (OI-Veh), GalNAc-Apc001 25 mg / kg, and sclerostin antibody 25 mg / kg. Wild-type mice were subcutaneously injected with PBS (WT-Veh) (once a week for 12 weeks). Col1a2 + / G610C Mice were sacrificed before OI-baseline (OI-BS) treatment.

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[0243] Sequence information

Claims

1. An aptamer conjugate comprising i) an aptamer that specifically binds to sclerostin, and ii) a specific ligand for E3 ubiquitin ligase (E3) and / or a specific ligand for asialoglycoprotein receptor (ASGPR).

2. The aptamer conjugate of claim 1, wherein the aptamer that specifically binds to sclerostin comprises i) a nucleotide sequence that is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to any one of SEQ ID NOs: 1-17; or ii) at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of any one of SEQ ID NOs: 1-17; or iii) a nucleotide sequence of any one of SEQ ID NOs: 1-17, preferably, a nucleotide sequence of SEQ ID NO:

17.

3. The aptamer conjugate of claim 1 or 2, wherein the aptamer that specifically binds to sclerostin has a Kd (dissociation constant) for sclerostin of less than 100 nM, preferably less than 50 nM, preferably less than 40 nM, preferably less than 30 nM, preferably less than 20 nM, preferably less than 10 nM or less.

4. The aptamer conjugate of any one of claims 1 to 3, wherein the aptamer that specifically binds to sclerostin is a modified aptamer, and the modified aptamer may comprise one or more modifications that impart enhanced nuclease resistance to the aptamer and / or modifications that extend the in vivo half-life of the aptamer.

5. The aptamer conjugate of claim 4, wherein the modification comprises a 3' inverted deoxythymidine (3'idT) modification.

6. The aptamer conjugate of claim 4, wherein the modification comprises replacing one or more naturally occurring nucleotides with modified nucleotides selected from 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and / or 2'propenyloxy modified nucleotides, preferably 2'-methoxy modified nucleotides.

7. The aptamer conjugate of claim 4, wherein the modification comprises an internucleotide modification, such as an internucleotide phosphorothioate bond modification.

8. The aptamer conjugate of claim 4, wherein the aptamer comprises a 2'-methoxy (2'-OMe) modification and / or a 3' inverted deoxythymidine (3'idT) modification.

9. The aptamer conjugate of any one of claims 1 to 8, wherein the aptamer is further conjugated to a fatty acid.

10. The aptamer conjugate of claim 9, wherein the fatty acid is selected from palmitic acid (PA), dodecanedioic acid (DA), tetradecanedioic acid, hexadecanedioic acid, stearic acid (SA), octadecanedioic acid, lauric acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA), preferably, the fatty acid is octadecanedioic acid.

11. The aptamer conjugate of any one of claims 1 to 10, wherein the E3-specific ligand is selected from von Hippel-Landau (VHL), murine double minute 2 (MDM2) and cereblon (CRBN); preferably, the E3-specific ligand is VHL, for example, the VHL comprises a structure shown in the following formula: 12 . The aptamer conjugate of claim 1 , wherein the E3-specific ligand is conjugated to the aptamer via a linker, for example, to the 5′ end of the aptamer.

13. The aptamer conjugate of claim 12, wherein the linker is a bifunctional linker, for example, the bifunctional linker comprises one of the structures shown below: ;or The bifunctional joint comprises the structure shown below:

14. The aptamer conjugate of claim 13, wherein the aptamer conjugate comprises the following structure: (FA / E3 ligand)-bifunctional linker-aptamer nucleotide sequence (5'-3'), wherein FA represents fatty acid, E3 ligand represents E3-specific ligand, wherein FA and E3 ligand are conjugated to the 5' end of the aptamer nucleotide sequence via a bifunctional linker, preferably, the fatty acid is octadecanedioic acid and / or the E3-specific ligand is VHL.

15. The aptamer conjugate of claim 14, wherein the aptamer conjugate comprises a structure represented by the following formula:

16. The aptamer conjugate of any one of claims 1 to 10, wherein the specific ligand of the asialoglycoprotein receptor (ASGPR) is N-acetylgalactosamine (GalNAc).

17. The aptamer conjugate of any one of claims 1 to 16, wherein the ASGPR-specific ligand, such as GalNAc, is conjugated to the aptamer via a linker.

18. The aptamer conjugate of claim 16 or 17, wherein the aptamer conjugate comprises a structure represented by the following formula: in 19. The aptamer conjugate according to any one of claims 16 to 18, wherein the aptamer conjugate comprises a structure represented by the following formula:

20. The aptamer conjugate of any one of claims 1 to 19, wherein the aptamer nucleotide sequence (5'-3' direction) is C(OMe)G(OMe)G(OMe)G(OMe)GTGTGGGTTCGTCGTTAGCTTGATTTGGCAGCU(OMe)G(OMe)C(OMe)C(OMe)-idT, wherein (OMe) represents a 2'-methoxy (2'-OMe) modification of the corresponding nucleotide and idT represents a 3' inverted deoxythymidine modification.

21. The aptamer conjugate according to any one of claims 1 to 20, wherein the aptamer conjugate is used for targeted degradation of sclerostin. The aptamer conjugate of claim 21 , wherein the aptamer conjugate is used for targeted degradation of sclerostin in cells.

23. The aptamer conjugate of claim 22, wherein the cell is a cancer cell, for example, the cancer cell is a cancer cell that highly expresses sclerostin, or the cancer cell is a cancer cell that highly expresses sclerostin and asialoglycoprotein receptor (ASGPR).

24. The aptamer conjugate of claim 23, wherein the cancer cell is a breast cancer cell (preferably a triple-negative breast cancer cell) or a liver cancer cell.

25. A method for treating a sclerostin-related disease, comprising administering a therapeutically effective amount of the aptamer conjugate of any one of claims 1 to 20 to a subject in need thereof, such as a human.

26. The method of claim 25, wherein the sclerostin-related disease is selected from the group consisting of osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), avascular necrosis, rheumatoid arthritis, bone fractures, osteoarthritis, myeloma, hypophosphatemic rickets, liver cancer, and triple-negative breast cancer.

27. A pharmaceutical composition comprising at least one aptamer conjugate according to any one of claims 1 to 20, and a pharmaceutically acceptable carrier or excipient.

28. Use of the aptamer conjugate according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 27 in the preparation of a medicament, wherein the medicament is used to treat a sclerostin-related disease.

29. The use of claim 28, wherein the sclerostin-related disease is selected from osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), ischemic osteonecrosis, rheumatoid arthritis, bone fractures, osteoarthritis, myeloma, hypophosphatemic rickets, liver cancer and triple-negative breast cancer.

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