WO2020163974A1 - Diagnostic use of aptamer for sclerostin - Google Patents

Abstract

An aptamer and an application thereof in diagnosing sclerostin-related diseases; the aptamer comprises a nucleotide sequence having at least about 90% homology, at least about 91% homology, at least about 92% homology, at least about 93% homology, at least about 94% homology, or at least about 95% homology with any one of SEQ ID NOs:1-17, or comprises at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides from any one of SEQ ID NOs: 1-17. The aptamer specifically binds to sclerostin.

Description

Diagnostic use of aptamers for osteosclerosin Technical field

The invention relates to the field of molecular diagnostics. Specifically, the present invention relates to the diagnostic use of aptamers for sclerostin.

Background of the invention

Aptamers are short single-stranded oligonucleotides that bind to their targets through conformational complementation (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Compared with antibodies, aptamers have similar affinity and specificity, but have some important advantages. For immunogenicity, aptamers are not recognized as foreign by the immune system, and do not stimulate negative immune responses because of their low molecular weight (Keefe, Pai et al., 2010). In terms of production and cost, aptamers can be identified in vitro under various selection conditions and can be easily synthesized by chemical methods, so production costs are lower and risks are lower (Banerjee, 2010). In terms of stability, aptamers have an infinite shelf life because they are not temperature sensitive and do not have any special cooling requirements during transportation, so there is no need for a continuous cold chain (Jayasena, 1999).

Osteosclerostin is a negative regulator of bone formation (Sclerostin), which inhibits the Wnt signaling pathway. Oste sclerostin has become a biomarker of bone metabolism, and its expression in vivo is associated with many diseases. It is expected in the art to develop aptamers for osteosclerosin for detecting osteosclerosin and/or diagnosing osteosclerosin-related diseases.

Brief description of the invention

In one aspect, the present invention provides a method for detecting the presence and/or the expression level of bone sclerostin in a biological sample, comprising:

a) Under the condition that a complex can be formed between the aptamer and the sclerostin, the biological sample and the control sample are respectively contacted with the aptamer for the sclerostin;

b) detect the formation of complexes,

Wherein the difference in the formation of the complex between the biological sample and the control sample indicates the presence of bone sclerostin and/or the expression level of bone sclerostin in the sample,

Aptamer

i) Contains at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity with any of SEQ ID NO: 1-17 Or a nucleotide sequence that is at least about 95% identical, or,

ii) comprising at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides in any one of SEQ ID NO: 1-17,

Preferably, the aptamer comprises the sequence of any one of SEQ ID NO: 1-17 or 19-24,

The aptamer specifically binds to bone sclerostin.

In some embodiments, the aptamer has a K _d for osteosclerosin 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.

In some embodiments, the aptamer for osteosclerosin is conjugated with fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, or tags that can be used for detection.

In some embodiments, the aptamer further comprises one or more modifications that confer enhanced nuclease resistance to the aptamer and/or enhance the half-life of the aptamer in vivo. In some embodiments, the modification includes a 3' inverse deoxythymidine (3'idT) modification. In some embodiments, the modification includes replacing one or more naturally occurring nucleotides with modified nucleotides selected from the group consisting of 2'-fluoro, 2'-methoxyethyl , 2'-methoxy or 2'propenoxy modified nucleotides, preferably 2'-methoxy modified nucleotides. In some embodiments, the modification includes an internucleotide modification, such as an internucleotide phosphorothioate bond modification. In some embodiments, the modification includes PEG modification. In some embodiments, the aptamer comprises 2'-methoxy (2'-OMe) modification, 3'inverted deoxythymidine (3'idT) modification and/or PEG modification.

In another aspect, the present invention also provides a method for diagnosing a disease related to bone sclerostin in a subject, the method comprising using the aptamer for sclerostin as defined above to detect a patient suspected of having a disease related to bone sclerostin. The presence of bone sclerostin and/or the expression level of bone sclerostin in the biological sample represents the existence and/or state of bone sclerostin-related diseases.

In another aspect, the present invention also provides a method for diagnosing osteosclerosin-related diseases in a subject by administering an aptamer for osteosclerosin as defined above to a subject suspected of suffering from osteosclerosin-related diseases, and then The accumulation of the aptamer in the subject is detected, thereby diagnosing a disease related to osteosclerosis. In some embodiments, detecting the accumulation of the aptamer in the subject is achieved by imaging the subject, the aptamer being labeled with an isotope, for example.

In one aspect, the present invention provides the use of the above-defined aptamer for sclerostin in preparing a kit for detecting the presence of sclerostin in a biological sample and/or the expression level of sclerostin.

In another aspect, the present invention also provides the use of the aptamer for osteosclerosin as defined above in the preparation of a kit for diagnosing osteosclerosis-related diseases in a subject.

In some embodiments of various aspects of the present invention, the bone sclerostin-related disease is, for example, a disease in which the expression level of bone sclerostin in a subject is abnormal.

In another aspect, the present invention provides a kit for use in the detection method and/or diagnosis method of the present invention, the kit comprising the above-defined aptamer for osteosclerosis, for detecting the The presence and/or amount of the aptamer for osteosclerosin, and instructions for use.

Brief description of the drawings

Figure 1 shows the enrichment of high-affinity aptamers for sclerostin by SELEX. (A) The binding affinity of the enriched ssDNA and unselected libraries to sclerostin. (B) The binding affinity of the enriched ssDNA library and the unselected library to the control protein.

Figure 2 shows the specific characterization of aptamer candidates. Compared with binding to hepatocytes and PBMC, the aptamer candidates show high selectivity for human osteosclerosis.

Figure 3 shows the affinity of the aptamer candidates identified from the ssDNA library with a 40 nt random region to recombinant human sclerostin. The dissociation constant (Kd) of aptamer candidates and antibodies to osteosclerosin was calculated by nonlinear curve fitting analysis. The Kd values of each aptamer candidate for bone sclerostin are: aptscl 6 is 4.2 nM, aptscl 9 is 3.4 nM, aptscl 15 is 45.6 nM, aptscl 46 is 43.1 nM, aptscl 56 is 43.1 nM and aptscl 132 is 42.2 nM. . The Kd value of the anti-sclerosin antibody is 3.55 nM.

Figure 4 shows the binding affinity of aptamer candidates identified from the ssDNA library with 25 nt random regions to recombinant human osteosclerosis. The dissociation constant (Kd) of each candidate was calculated by nonlinear curve fitting analysis. The Kd values of each aptamer candidate and antibody are: aptscl 32 is 0.18 nM, aptscl 29 is 0.28 nM, aptscl 22 is 0.76 nM, aptscl 16 is 0.22, aptscl 3 is 0.04 nM, aptscl 2 is 0.006 nM, aptscl 1 It is 0.02nM. The Kd value of the anti-sclerosin antibody is 3.55 nM.

Figure 5 shows the use of TOP-Wnt-induced luciferase reporter gene assay to evaluate the inhibitory ability of aptamer candidates and antibodies. (A) Compared with antibodies, Wnt signaling-mediated luciferase activity in MC3T3-E1 cells treated with aptamer candidates. Aptscl 56, aptscl 6, aptscl 3 and anti-oste sclerostin antibodies can effectively inhibit the antagonistic effect of bone sclerostin on Wnt signaling and release Wnt-induced luciferase activity. When the concentration of aptscl 56 and aptscl 6 reached 25μg/ml and 47.4μg/ml, the response reached a stable level. When treated with antibodies, the response was still not stable when the concentration was increased to 20 mg/ml in this experiment. (B) Analysis of the inhibitory ability of aptscl 56, the EC50 of aptscl 56 is 19.7 μg/ml. (C) Analysis of the inhibitory ability of aptscl 6, the EC50 of aptscl 6 is 36.8 μg/ml. (D) Analysis of the inhibition ability of aptscl 3, the EC50 of aptscl 3 is 18.2μg/ml.

Figure 6 shows the characterization of the binding of truncated aptscl3 to bone sclerostin. (A) aptscl3-1, -2, -3, -4, and -5 maintain high affinity for sclerostin, while aptscl3-6 shows low binding ability to sclerostin and cannot fit the affinity analysis curve; (B) cut The short aptscl3-5 still maintains a high binding affinity and retains a high inhibitory effect (EC50=28.4μg/ml) on the Wnt signaling pathway in the antagonistic cells of bone sclerostin.

Figure 7 shows the serum stability assessment of modified aptscl56 compared to unmodified aptscl56. All aptamers were treated with 10% and 100% mouse serum for 0 to 72 hours. After 48 hours of incubation in 10% mouse serum, almost all unmodified aptscl56 was degraded. 2'-OMe and 3'-idT modified aptscl56 can be maintained in 10% mouse serum for 48 hours. In 100% serum, unmodified aptscl56 was rapidly and completely degraded after 8 hours; at 72 hours, a small amount of modified aptamers remained.

Figure 8 shows the serum stability assessment of modified aptscl3-5 compared to unmodified aptscl3-5. All aptamers were treated with 10% and 100% mouse serum for 0 to 72 hours. After 24 hours of incubation in 10% mouse serum, Aptscl3-5 was degraded. 2'-OMe and 3'-idT modified aptscl3-5 can be maintained in 10% mouse serum for 48 hours. In 100% serum, unmodified aptscl3-5 was completely degraded rapidly after 8 hours, while modified aptscl3-5 remained intact after 72 hours.

Figure 9 shows the affinity and inhibitory efficacy of chemically modified aptscl56 and aptscl3-5. After chemical modification, both aptscl56 and aptscl3-5 maintained high affinity to osteosclerosis and in vitro inhibitory efficacy.

Figure 10 shows the pharmacokinetics of a single subcutaneous injection of Aptscl56 in 6 rats: the pharmacokinetic curve fitted by the software DAS (left) and the actual pharmacokinetic curve (right).

Figure 11 shows the pharmacokinetics of a single subcutaneous injection of PEG40K-aptscl56 in 6 rats: the pharmacokinetic curve fitted by the software DAS (left) and the actual pharmacokinetic curve (right).

Figure 12 shows the pharmacokinetics of a single subcutaneous injection of Aptscl56 and PEG40K-aptscl56 in rats.

Detailed description of the invention

Unless otherwise indicated or defined, all terms used have their usual meanings in the art, and this meaning will be understood by those skilled in the art. Refer 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 general existing references in this article Technology; In addition, unless otherwise specified, all methods, steps, technologies and operations not specifically detailed can be and have been performed in a manner known per se, which will be understood by those skilled in the art. Also refer to, for example, the standard manual, the above-mentioned general prior art and other references cited therein.

definition

As used herein, the term "nucleotide" refers to ribonucleotides or deoxyribonucleotides, or modified forms and analogs thereof. Nucleotides include species, including purines (for example, adenine, hypoxanthine, guanine and their derivatives and analogs) and pyrimidines (for example, cytosine, uracil, thymine, and their derivatives and analogs)物).

In this article, "nucleic acid", "oligonucleotide" and "polynucleotide" are used interchangeably to refer to polymers of nucleotides, and include DNA, RNA, DNA/RNA hybrids and these types of nucleic acids, The modification of oligonucleotides and polynucleotides includes appending various entities or parts to any position of the nucleotide unit. The terms "polynucleotide", "oligonucleotide" and "nucleic acid" include double or single stranded molecules. Nucleic acid, oligonucleotide and polynucleotide are broader terms than the term aptamer, so the terms nucleic acid, oligonucleotide and polynucleotide include but are not limited to aptamer.

As used herein, "aptamer" refers to a non-naturally occurring nucleic acid that has a desired effect on a target molecule. The desired effects include, but are not limited to, binding to the target, catalytically changing the target, reacting with the target in a manner that modifies or alters the target or the functional activity of the target, covalently connecting the target and promoting the target The reaction between the target and other molecules. In some embodiments, the effect is specific binding affinity for a target molecule (such as osteosclerosin), such a target molecule is a three-dimensional chemical structure rather than a polynucleotide, which does not rely on Watson/Crick base pairing. Or the mechanism of triple helix formation to bind the aptamer, wherein the aptamer is not a nucleic acid with a known physiological function bound by the target molecule. In this context, the "specific binding" of an aptamer to its target (such as osteosclerosin) means that the aptamer usually binds with a much higher affinity than other non-target components in the mixture or sample. Binding to its target.

Sequence "identity" has the art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using disclosed 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 the skilled person (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ). Many algorithms can be used to determine the percent sequence identity. An example of an algorithm suitable for determining the 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. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (hereinafter "NCBI"). The 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.

The term "biological sample" refers to any material, solution or mixture obtained from an organism. This includes blood (including whole blood, white blood cells, peripheral blood mononuclear cells, plasma, and serum), sputum, exhalation, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph, nipple aspiration fluid, Bronchial aspiration fluid, synovial fluid, joint aspiration fluid, cells, cell extracts and cerebrospinal fluid. This also includes the experimentally separated parts of all the aforementioned substances. The term "biological sample" also includes, for example, materials, solutions or mixtures containing homogenized solid materials (such as from stool samples, tissue samples, or tissue biopsies). The term "biological sample" also includes materials, solutions or mixtures derived from cell lines, tissue cultures, cell cultures, bacterial cultures, virus cultures, or cell-free biological systems.

Aptamer for sclerostin

Based on the protein-SELEX technology, the present inventors used osteosclerosin as the target protein for positive screening, and used unrelated proteins for negative screening, and finally selected aptamers that specifically bind to osteosclerosin with high affinity. The sclerostin described herein is preferably human sclerostin, for example, the sclerostin whose amino acid sequence is shown in SEQ ID NO: 18.

Exemplary human sclerostin amino acid sequence:

Therefore, in one aspect, the present invention provides an aptamer for osteosclerosin, the aptamer comprising at least about 90% identity, at least about 91% identity, at least about 90% identity to any of SEQ ID NO: 1-17 About 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identical nucleotide sequence, or the aptamer comprises at least 30, at least 35, at least 40, at least 45, at least 50, or any of SEQ ID NO: 1-17 More consecutive nucleotides. In some embodiments, the aptamer specifically binds bone sclerostin. In some preferred embodiments, the aptamer comprises the nucleotide sequence of any one of SEQ ID NO: 1-17 and 19-24, more preferably, the aptamer comprises SEQ ID NO: 1, 3, The nucleotide sequence of one of 10 or 19-23.

In some embodiments, the aptamer of the present invention has 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 K _d (dissociation constant ). The K _{d is} measured by, for example, an enzyme-linked oligonucleotide assay (ELONA).

In some embodiments, the aptamer of the present invention may also include one or more modifications. For example, the modification is a modification that confers enhanced nuclease resistance to the aptamer and/or enhances the half-life of the aptamer in vivo.

The modification includes, for example, 3'and 5'modification, such as 3'and 5'capping. In some embodiments, the aptamer is capped with inverted deoxythymidine at the 3'end, that is, 3'inverted deoxythymidine (3'idT) is modified.

The modification may also include the replacement of one or more naturally occurring nucleotides with modified nucleotides. For example, the modified nucleotides include, but are not limited to, 2'-fluoro, 2'-methoxyethyl, 2'-methoxy and/or 2'propyleneoxy modified nucleotides (ie, ribose The 2'-position hydroxyl group is substituted by fluorine, methoxyethyl, methoxy or propyleneoxy, etc.). The modified nucleotides may 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, refer to International Patent Application WO 2011/130195 and its cited documents. In some preferred embodiments, the modification is a 2'-methoxy (2'-OMe) modification. In some embodiments, one or more, such as 4 nucleotides, at the 5'and/or 3'end of the aptamer are modified as indicated, such as 2'-methoxy (2'-OMe) modification.

The modifications also include internucleotide modifications, such as those with uncharged bonds (such as methyl phosphonate, phosphotriester, phosphoamine ester, carbamate, etc.) and those with Internucleotide modification of charged bonds (such as phosphorothioate, phosphorodithioate, etc.), internucleotide modification of intercalating agents (such as acridine, psoralen, etc.), containing chelating agents (such as Metals, radioactive metals, boron, oxidizing metals, etc.) internucleotide modifications, internucleotide modifications containing alkylating agents, and internucleotide modifications with modified bonds (for example, alpha anomeric nucleic acid, etc.).

The modification may also include pegylation modification (PEG modification). Conjugation with PEG can extend the half-life of the aptamer. In some embodiments, the molecular weight of the PEG is about 1 kDa to about 100 kDa, for example, about 10 kDa to about 80 kDa, about 20 kDa to about 60 kDa, about 30 kDa to about 50 kDa, about 40 kDa. In some embodiments, the PEG may be conjugated to the 5' end of the aptamer. In some embodiments, the PEG may be conjugated to the 3' end of the aptamer.

In some embodiments, the aptamer may comprise a combination of various modifications described above. For example, the aptamer may include 2'-methoxy (2'-OMe) modification, 3'inverse deoxythymidine (3'idT) modification and/or PEG modification. Preferably, the molecular weight of the PEG is about 40 kDa.

Detection of bone sclerostin

In another aspect, based on the highly specific binding of the aptamer of the present invention to bone sclerostin, the present invention also provides a method for detecting the presence of sclerostin and/or the expression level of sclerostin in a biological sample, including :

a) Under the condition that a complex can be formed between the aptamer for bone sclerostin of the present invention and the bone sclerostin, the biological sample and the control sample are respectively contacted with the aptamer for bone sclerostin of the present invention;

b) detect the formation of complexes,

Wherein the difference in the formation of the complex between the biological sample and the control sample indicates the presence of osteosclerosin and/or the expression level of osteosclerosin in the sample. The selection of the control sample belongs to the routine skills of those skilled in the art and can be determined according to actual needs. The biological sample can be selected from serum, plasma, whole blood, synovial fluid and the like.

In some embodiments, the aptamer is conjugated with fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, tags, etc. that can be used for detection.

Many aptamer-based target determination methods are known in the art, and these methods can be modified and applied to the present invention.

The present invention therefore also covers the use of the aptamer for osteosclerosin of the present invention in the preparation of a kit for detecting the presence of osteosclerosin in a biological sample and/or the expression level of osteosclerosin.

Disease diagnosis

In another aspect, the present invention also provides the use of the aptamer for sclerostin of the present invention in preparing a kit for diagnosing a disease related to sclerostin in a subject. In some embodiments, the bone sclerostin-related disease is, for example, a disease in which the expression level of bone sclerostin is abnormal in the subject.

In another aspect, the present invention also provides a method for diagnosing a disease related to bone sclerostin in a subject, the method comprising using the aptamer for sclerostin of the present invention to detect organisms from a subject suspected of having a disease related to bone sclerostin. The presence of bone sclerostin and/or the expression level of bone sclerostin in the scientific sample, the presence of bone sclerostin and/or the expression level of bone sclerostin represent the existence and/or state of bone sclerostin-related diseases. In some embodiments, the bone sclerostin-related disease is, for example, a disease in which the expression level of bone sclerostin is abnormal in the subject.

In another aspect, the present invention also provides a method for diagnosing a disease related to bone sclerostin in a subject by administering the aptamer for sclerostin of the present invention to a subject suspected of having a disease related to sclerostin, and then detecting Accumulation of the aptamer in the subject, thereby diagnosing osteosclerosis-related diseases. In some embodiments, detecting the accumulation of the aptamer in the subject is achieved by imaging the subject, the aptamer being labeled with an isotope, for example. In some embodiments, the bone sclerostin-related disease is, for example, a disease in which the expression level of bone sclerostin is abnormal in the subject.

As used herein, "oste sclerostin-related 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 (bone Necrosis), fracture and implant healing (dental implants and hip implants), bone loss due to other conditions (for example, related to HIV infection, cancer, and arthritis). Other "sclerosin-related diseases" include, but are not limited to: rheumatoid arthritis, osteoarthritis, arthritis, and osteolytic lesions.

As used herein, "oste sclerostin-related diseases" include bone sclerostin-related cancers, such as myeloma (e.g., multiple myeloma with osteolytic lesions), breast cancer, colon cancer, melanoma, hepatocellular carcinoma, Epithelial cancer, esophageal cancer, brain cancer, lung cancer, prostate cancer or pancreatic cancer, and any metastases thereof.

"Sclerosingrin-related diseases" may also include kidney disease and cardiovascular disease caused by at least the expression of osteosclerosin in the kidney and in the cardiovascular system. The conditions include, but are not limited to, nephropathy such as: glomerular diseases (eg, acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, and Systemic diseases such as systemic lupus erythematosus, Goodpasture’s syndrome, multiple myeloma, diabetes, polycystic kidney disease, neoplasia, sickle cell disease, and chronic inflammation and other systemic diseases related to glomerular damage), renal tubules Diseases (for example, acute tubular necrosis and acute renal failure, polycystic kidney disease, medullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and tubular acidosis), tubulointerstitial diseases (for example, pyelonephritis) , Drug and toxin-induced tubulointerstitial nephritis, hypercalcemia nephropathy, and hypokalemic nephropathy), acute and rapidly progressive renal failure, chronic renal failure, kidney stones, gout, vascular diseases (for example, hypertension and Nephrosclerosis, microangiopathic hemolytic anemia, atherosclerotic embolism nephropathy, necrosis of the diffuse cortex, and renal infarction), or tumors (eg, renal cell carcinoma and nephroblastoma).

The diseases also include, but are not limited to, cardiovascular diseases such as: ischemic heart disease (for example, angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, pulmonary heart disease, valvular heart disease (for example, , Rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (for example, valve and blood vessel occlusion damage, atrioventricular septal defect, and long-standing Arterial duct), or cardiomyopathy (for example, myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy).

The subject can be any animal (domestic, domestic or wild), including but not limited to cats, dogs, horses, pigs, and cows, and is preferably human subjects. As used herein, the terms patient, individual, and subject can be used interchangeably.

Reagent test kit

The scope of the present invention also includes a kit used in the detection method and/or diagnosis method of the present invention, which includes the aptamer for osteosclerosin of the present invention, and instructions for use. The kit may further include at least one additional detection reagent, which is used to detect the presence and/or amount of the aptamer for bone sclerostin of the present invention. The kit generally includes a label indicating the intended use and/or method of use of the contents of the kit, that is, instructions for use. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

Example

The following will further illustrate the present invention by way of examples, but the present invention is not limited to the scope of the described examples.

Example 1. Enrichment and screening of high-affinity aptamers for bone sclerostin by SELEX

The sequence in the ssDNA library consists of 18 nt conserved regions at both ends and a central random region. Two ssDNA libraries with random sequences of different lengths were used. The sequence in the long ssDNA library contains a 40 nt random region (5'-CGTACGGTCGACGCTAGC-(N) ₄₀ -CACGTGGAGCTCGGATCC-3'), and the sequence in the short ssDNA library contains a ₂₅ nt random region (5'-CGTACGGTCGACGCTAGC-(N) ₂₅ -CACGTGGAGCTCGGATCC- 3'). Synthetic forward primer (FP: 5'-CGTACGGTCGACGCTAGC-3') and biotinylated reverse primer (Bio-RP: 5'-Biotin-GGATCCGAGCTCCACGTG-3') were used to amplify ssDNA during selection. All oligonucleotides were purified by HPLC after synthesis.

The protein SELEX method was performed to identify high-affinity aptamers (Ellington and Szostak, 1990; Tuerk and Gold, 1990). 100-30 picomoles of His _6- labeled osteosclerosin were immobilized on NTA magnetic beads at 4°C for 1 hour (Murphy, Fuller et al., 2003). The 1 nanomolar ssDNA library was denatured at 95°C for 5 minutes and quickly cooled to 4°C, and then incubated with immobilized bone sclerostin at room temperature for 0.5-1 hour. Remove unbound sequences with wash buffer. After washing, the bound DNA-protein-NTA was collected, resuspended in H ₂ O/Tween 20, and applied to PCR amplification. Use unmodified forward primers and biotinylated reverse primers for PCR amplification (Step 1: 95°C initial denaturation for 1 minute; Step 2: 95°C denaturation for 30 seconds, 56°C annealing for 30 seconds, and 72°C extension for 30 seconds , Repeat 12 cycles; and step 3: 72°C and finally extend for 5 minutes). The PCR product is applied to streptavidin magnetic beads through biotin-streptavidin binding. The single-stranded sequence was regenerated by treatment with 0.2M NaOH. Negative selection is made for other His _6- labeled irrelevant proteins immobilized on NTA magnetic beads. A total of 20 rounds of SELEX were performed for each option. The DNA pool from the last round was subjected to high-throughput second-generation sequencing (NGS).

Figure 1 shows that the affinity of the DNA pool to sclerostin increases after the 10th and 20th rounds of selection, indicating that high-affinity sclerostin aptamers are enriched by SELEX.

Example 2. Specific identification of candidate osteosclerosis aptamers

According to the NGS results, representative aptamers with high frequency of occurrence were synthesized for specific determination. The detailed sequences of these aptamer candidates are listed in Table 1.

In order to determine the specificity of aptamer candidates to osteosclerosin, representative aptamer candidates and random sequence (RS) (negative control) were synthesized using N-terminal biotinylation modification. The specificity of each aptamer/RS to osteosclerosin was determined by enzyme-linked oligonucleotide assay (ELONA) using 1 μM. 160 ng of purified recombinant human sclerostin was coated in a 96-well microtiter plate by incubating in 100 μl PBS at 4° C. overnight. Then the plate was blocked with blocking buffer (PBS, 0.1 % Tween 20 and 1% BSA) for 1 hour at room temperature, and washed with SELEX B&W buffer (PBS, 1 mM MgCl ₂ , 0.1% Tween 20 and 0.1% BSA) 4 Times. The aptamer candidate was denatured at 95°C for 10 minutes and quickly cooled on ice for 10 minutes before use. Add 1μM biotin-labeled aptamer to each well, then add SELEX B&W buffer to 100μl, incubate at room temperature for 45min, and continue to shake gently. After binding, the plate was washed 4 times with SELEX B&W buffer to remove non-specific and very weak binding, and then washed 4 times with PBST+0.1% BSA. Add 100μl streptavidin-HRP/goat anti-human IgG Fc-HRP (diluted 1:10000 in PBST+0.1%BSA) to each well and incubate for 30/60 minutes, and wash with PBST+0.1%BSA4 Times. Add 50 μl TMB to each well and incubate for 20 minutes. The reaction was stopped by adding 50 μl 2M H ₂ SO ₄ . The absorbance at 450nm was measured with a microplate reader (Stoltenburg, Krafcikova et al., 2016). In order to determine the binding ability of aptamer candidates to hepatocytes/PBMC, the characterization procedure is similar to ELONA. 300,000 cells were incubated with each aptamer candidate, washed and separated by centrifugation.

The results showed that the aptamer candidates identified from the long and short ssDNA libraries showed high selectivity to human sclerostin when compared with the binding of hepatocytes and PBMC (Figure 2). Aptamer candidates aptscl 6, 9, 15, 27, 34, 36, 46, 51, 56, 132, and 140 identified from the long ssDNA library, and aptscl 1, 2, 3, 5, 8 identified from the short ssDNA library , 12, 16, 22, 29, and 32, showing high binding specificity to osteosclerosin, so they were selected for the following affinity characterization.

Example 3. Affinity identification of candidate osteosclerosis aptamers

An enzyme-linked oligonucleotide assay (ELONA) was performed to determine the binding affinity of the aptamer candidate to osteosclerosin (Drolet, Moon-McDermott et al., 1996). Similarly, an enzyme-linked immunosorbent assay (ELISA) was performed to determine the binding affinity of anti-sclerosin antibody (Romosozumab, a humanized therapeutic antibody against bone sclerostin, purchased from Creative Biolabs) to human sclerostin (Engvall and Perlmann , 1971). 160 ng of purified recombinant human sclerostin was coated in a 96-well microtiter plate by incubating in 100 μl PBS at 4° C. overnight. Then the plate was blocked with blocking buffer (PBS, 0.1 % Tween 20 and 1% BSA) for 1 hour at room temperature, and washed with SELEX B&W buffer (PBS, 1 mM MgCl ₂ , 0.1% Tween 20 and 0.1% BSA) 4 Times. The aptamer candidate was denatured at 95°C for 10 minutes and quickly cooled on ice for 10 minutes before use. Add the appropriate concentration of biotinylated aptamer/antibody to each well, then add SELEX B&W buffer to 100 μl, and incubate at room temperature for 45 minutes with continuous gentle shaking. After binding, the plate was washed 4 times with SELEX B&W buffer to remove non-specific and very weak binding, and then washed 4 times with PBST+0.1% BSA. Add 100μl streptavidin-HRP/goat anti-human IgG Fc-HRP (diluted 1:10000 in PBST+0.1%BSA) to each well and incubate for 30/60 minutes, and wash with PBST+0.1%BSA4 Times. Add 50 μl TMB to each well and incubate for 20 minutes. The reaction was stopped by adding 50 μl 2M H ₂ SO ₄ . The absorbance at 450nm was measured with a microplate reader (Stoltenburg, Krafcikova et al., 2016). The data was analyzed with Origin software (OriginLab, Northampton, MA). Using the nonlinear curve fitting model Hyperbl to draw the binding curve, the Hyperbl model equation is y=P1*x/(P2+x), and P2 is the Kd value.

For the aptamer candidates identified from the ssDNA library containing 40 nt random regions, aptscl 6, 9, 15, 46, 56 and 132 have high affinity for bone sclerostin and have nanomolar dissociation constant (Kd) values (Kd The values were 4.2, 3.4, 45.6, 43.1 and 42.2 nM) (Figure 3). But aptscl 36, 140, 136 and random sequence (RS) failed to fit. For the aptamer candidates identified from the ssDNA library containing 25 nt random regions, aptscl 32, 29, 22, 16, 3, 2, and 1 have higher binding affinity to sclerostin, with Kd values of 0.18, 0.28, and 0.76, respectively , 0.02, 0.04, 0.006 and 0.02nM (Figure 4). The random sequence shows low binding capacity to osteosclerosin and cannot be fitted. In contrast, the Kd value of anti-sclerosin antibody to osteosclerosin is 3.55 nM.

The high-affinity aptamer is particularly suitable for detecting bone sclerostin in biological samples and for diagnosing bone sclerostin-related diseases in vivo or in vitro.

Example 4. In vitro evaluation of the inhibitory ability of the candidate osteosclerosin aptamer on the activity of osteosclerosis

Using TOP-Wnt-induced luciferase reporter gene assay in osteoblast MC3T3-E1 cells (van Bezooijen, Svensson et al., 2007; Shum, Chan et al., 2011) to study the inhibition of aptamer candidates The ability of bone sclerostin to antagonize Wnt signaling.

MC3T3-E1 cells were seeded in a 24-well plate, and on the second day, FuGENE HD transfection reagent (Promega) was used to transfect the corresponding reporter gene plasmid (100ng), Wnt3a plasmid (800ng) and bone sclerosis protein pellet (800ng). After 10 hours of transfection, the medium was replaced with fresh medium and the cells were treated with aptamer/antibody. After 24 hours of treatment, the cells in each well were lysed with 100 μl of passive lysis buffer, and 20 μl was taken for analysis. According to the manufacturer's protocol (Promega), luciferase assay reagent II and Stop&Glo reagent are automatically prepared through SpectraMax i3x Multi-Mode Detection Platform (Molecular Device), and the data is analyzed accordingly (Grentzmann, Ingram et al., 1998; McNabb ,Reed et al., 2005).

The results are shown in Figure 5, aptscl 56, aptscl 6, aptscl 3 and anti-oste sclerostin antibodies can effectively inhibit the antagonistic effect of bone sclerostin on Wnt signaling and release Wnt-induced luciferase activity. The inhibitory effect on bone sclerostin is dose-dependent. When the concentrations of aptscl 56 and aptscl6 reach 25 μg/ml and 47.4 μg/ml, the response is stable. When treated with antibodies, the response was still unstable when the concentration was increased to 20 mg/ml. In addition, the inhibition ability of aptscl56, aptscl 6 and aptscl 3 was analyzed by nonlinear curve fitting. The EC50 of Aptscl 56, aptscl 6, and aptscl 3 were 19.7 μg/ml, 36.8 μg/ml and 18.2 μg/ml, respectively.

Aptamers with high affinity but low inhibition may be more suitable for in vivo diagnostic purposes.

Table 1. Sequences of osteosclerosin aptamer candidates

Example 5 Truncation and characterization of aptscl3

Aptscl3, which showed high affinity for sclerostin and inhibitory potency, was truncated (Table 2). The same protocol as in the previous study was used to test the binding affinity and in vitro inhibition efficacy. aptscl3-1, aptscl3-2, aptscl3-3, aptscl3-4 and aptscl3-5 maintained high binding affinity to sclerostin, with Kd values of 0.86, 0.52, 0.2 and 0.22 nM, respectively. aptscl3-6 could not fit the binding curve in this concentration range, indicating that the binding capacity to sclerostin was low (Figure 6a). In addition, aptscl3-5 retained a high inhibitory effect on the antagonistic effect of sclerostin on Wnt signaling (EC50 = 28.4 μg/ml (Figure 6b)).

Table 2. Sequence truncation of the aptamer candidate aptscl3

Example 6. Evaluation of serum stability of chemically modified aptamer candidates

The inventors have selected a DNA aptamer for sclerostin, and finally developed two truncated aptamers, called aptscl56 and aptscl3-5, which specifically bind to sclerostin closely and have a low nanomolar range The dissociation constant within. 2'-O-methyl (2'-OMe) modifications of aptamers have previously been used as post-selection modifications because of their enhanced nuclease resistance and elevated duplex melting temperature, as in clinical trials Shown in (Fine, Martin et al. 2005; Gupta, Hirota et al. 2014). The 3'end capping of reverse dT is also a common strategy for aptamers used for disease treatment in ongoing or completed clinical trials (Padilla, Sousa et al. 1999; Ruckman, Green et al. 1998). Therefore, this example evaluated whether the serum stability of aptscl56 and aptscl3-5 can be improved by 2'-OMe and 3'-end reverse dT (3'-idT) modification.

experimental design:

The modified nucleotides are introduced during the synthesis process. The serum metabolic stability of modified and unmodified aptamers was evaluated in freshly prepared mouse serum. All aptamer samples were incubated with 10% and 100% mouse serum at 37°C for 0, 2, 4, 8, 12, 24, 36, 48, and 72 hours, respectively. At the specified time, the aptamer samples were quickly frozen in a dry ice bath and then stored at -80°C until all samples were harvested for evaluation. The stability of all aptamer samples is expressed as the band density of intact aptamers remaining after incubation, which can be determined by agarose gel electrophoresis.

DNA synthesis scheme:

Use commercially available 5'-O-DMT-2'-deoxynucleoside (ABz, CAc, GiBu and T) phosphoramidite monomer, 5'-O-DMT-2'-O-methyl nucleoside (ABz , CAc, GiBu and T) phosphoramidite monomer and/or 5'-O-DMT-2'-F-nucleoside (ABz, CAc, GiBu and T) phosphoramidite monomer in K&A H8 standard DNA/RNA Modified and unmodified DNA sequences were synthesized on a synthesizer on a 1 μmole scale (Beaucage and Caruthers 2001). The modified aptscl56 sequence is CGGG G TGTGG GTTCG TCGTT AGCTT GATTT GGCAG CT GCCC -idT, and the underlined nucleotide is a 2'-OMe modification. The modified sequence of aptscl3-5 is GCTA G CTGTT GTACA TCGCC TTACG CA CGT G -idT, and the underlined nucleotide is 2'-OMe modification.

Evaluation plan:

The band density of all aptamer samples was determined by a molecular imager (Bio-Rad) (Klussmann, Nolte et al. 1996, Siller-Matula, Merhi et al. 2012). The binding affinity and in vitro inhibition potency determinations were performed using the same protocol as in the previous study.

result:

For aptscl56, the unmodified aptamer was completely degraded after 48 hours in 10% serum, and remained in 100% serum for only 8 hours. 2'-OMe and 3'-idT modified aptscl56 were kept in 10% mouse serum for 72 hours, and degraded after 12 hours in 100% mouse serum. At 72 hours, a small amount of modified aptamers still remained (Figure 7).

For aptscl3-5, the unmodified aptamer was degraded 24 hours after incubation in 10% mouse serum. 2'-OMe and 3'-idT modified aptscl3-5 can be maintained in 10% mouse serum for 48 hours. In 100% serum, unmodified aptscl3-5 was rapidly and completely degraded after 8 hours, while modified aptscl3-5 maintained integrity after 72 hours (Figure 8).

The chemically modified aptscl56 and aptscl3-5 showed high binding affinity to bone sclerostin, with Kd values of 6.55 and 0.54 nM, respectively. In addition, chemically modified aptscl56 and aptscl3-5 can effectively relieve the inhibitory effect of bone sclerostin on Wnt signaling in cells, and their efficacies were 14 and 11 μg/ml, respectively (Figure 9).

in conclusion:

Modification with 2'-OMe and 3'-idT can further promote the development of aptscl56 and aptscl3-5 into therapeutic nuclease-resistant aptamers.

Example 7. PEG modified aptamer

The present invention determines the plasma pharmacokinetics of PEG-modified and non-PEG-modified aptamers (PEG40K-aptscl56 and aptscl56) for bone sclerostin after subcutaneous administration in rats. The aptscl56 sequence is CGGGG TTGTG GTTCG TCGTT AGCTT GATTT GGCAG CTGCCC-idT, and each nucleotide of the start CGGG and the end GCCC is modified with 2'-OMe. On this basis, PEG40K-aptscl56 is further connected to PEG40K (PEG with a molecular weight of 40,000) at the 5'-end.

experimental design:

The pharmacokinetic study of the aptamers aptscl56 and PEG40K-aptscl56 was performed in 6-month-old female primitive Sprague-Dawley rats, which were fed a standard laboratory diet ad libitum and reared under controlled conditions (12-hour photoperiod , 20°C). Rats were treated with 6.1 mg/kg aptscl56 and 25 mg/kg PEG40K-aptscl56 through a single subcutaneous injection. Aptscl56 and PEG40K-aptscl56 were dissolved in saline at concentrations of 1.6 mg/ml and 6.2 mg/ml, respectively (Judith M. Healy, Ryan M. Boomer et al., 2004). At different time points (aptscl56: 5min, 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h; PEG40K-aptscl56: 30min, 1h, 2h, 4h, 8h, 12h, 24h, 30h, 36h, 48h, 54h , 62h, 70h, 76h, 84h, 96h, 107h) blood samples were collected from repeated rats (n=6) in each group, and plasma was separated. After treatment with proteinase K, the remaining aptscl56 and PEG40K-aptscl56 in the plasma were quantified by HPLC.

Evaluation plan:

Sample preparation: Collect approximately 800μl of blood from each rat through the orbital vein, and collect it into a tube containing sodium-heparin as an anticoagulant (1.8ml vacuum container, BD Biosciences), and then immediately place it on wet ice (Healy , Lewis et al. 2004, Perschbacher, Smestad et al. 2015). The plasma was separated by centrifugation at 6000g for 10 minutes at 4°C within 1 hour after collection, and stored at -80°C until analysis (Healy, Lewis et al. 2004, Siller-Matula, Merhi et al. 2012, Gao, Shen et al.2016). Before analysis, 25μl digestion buffer (60mM Tris-HCl, pH8.0, 100mM EDTA and 0.5% SDS) and 75μl protease solution (1mg/mL proteinase K in 10mM Tris HCl, pH 7.5, 20mM CaCl ₂ , 10% glycerol) v/v) Add 50μl plasma sample. The samples were then incubated overnight at 55°C with shaking. After incubation, the samples were centrifuged (14000 rpm; 4°C; 15 minutes) and 100 μl of supernatant was removed and transferred to an HPLC injection vial (Siller-Matula, Merhi et al., 2012).

HPLC quantification: The HPLC system is equipped with a C4 column to quantify PEG40K-aptscl56 in plasma samples collected at different time points, and a C18 column is used to quantify aptscl56. This method uses a mobile phase elution gradient made of phase A (TEAA[pH 7.0]) and phase B (acetonitrile). When the temperature of the column oven is set at 50°C, the flow rate is 1.0 mL/min. The measured injection volume is 20uL. Standards were prepared in blank rat plasma containing different concentrations of aptscl56 and PEG40K-aptscl56 sodium-heparin (Gao, Shen et al., 2016). All reported concentrations of aptscl56 and PEG40K-aptscl56 are based on the quality of aptscl56. Calculate the aptamer concentration in the plasma sample according to the standard curve.

Pharmacokinetic analysis: Draw Aptscl56 and PEG40K-aptscl56 concentration versus time curves, and analyze each rat by software DAS 3.0 (BioGuider Co., Shanghai, China). Average the obtained pharmacokinetic parameters. The half-life (t1/2) of the aptamer was calculated based on the time required for the aptamer to eliminate half of the maximum plasma concentration (Grieken and Bruin 1994). According to the pharmacokinetic curve, the maximum plasma concentration (C max) and the maximum plasma concentration time (T max) were obtained. The area under the curve (AUC) is calculated from the start of drug administration, and ends when the concentration in plasma is negligible (Rowland, Benet et al. 1973, Toutain and Bousquet-Melou 2004, Siller-Matula, Merhi et al. 2012). Subsequently, the dosing interval of multiple doses of PEG40K-aptscl56 was calculated according to the following formula: D=1/1-e- ^Ke·t (Ke: elimination constant, Ke=ln2/T1/2; t: dosing interval; D: giving Drug ratio = loading dose/maintenance dose) (Birkett 1996, Jambhekar 2012).

result:

The lower limit of HPLC quantification of aptscl56 is 10ul/mL, and the linear concentration range is from 10μg/mL to 360ug/mL. The average elimination half-life (Elim.T1/2) of aptscl56 aptamer in Sprague-Dawley rats after subcutaneous administration was 1.8 hours. The average value of C max is 265.5 μg/ml, and T max is 0.5 hours (Figure 10, Table 3).

The lower limit of HPLC quantification of PEG40K-aptscl56 is 7.5μg/mL, and the linear concentration range is 7.5μg/mL to 240μg/mL. The average elimination half-life (Elim.T1/2) of PEG40K-aptscl56 in Sprague-Dawley rats after subcutaneous administration was 66.9 hours. The average value of C max is 152.8 μg/ml, and T max is 72 hours (Figure 11, Table 4).

Table 3. Pharmacokinetic parameters of aptscl56 administered subcutaneously

Table 4. Pharmacokinetic parameters of PEG40K-aptscl56 administered subcutaneously

discuss:

This example studied the pharmacokinetic characteristics of PEG40K-aptscl56 and aptscl56 administered subcutaneously in S.D. rats. Compared with the 1.8-hour elimination half-life of aptscl56, the half-life of PEG40K-aptscl56 was significantly extended by 65.1 hours. This indicates that PEG has a significant effect on prolonging the residence time of aptscl56 in vivo. However, the concentration of PEG40K-aptscl56 in plasma was lower than that of aptscl56 as a whole (Figure 12). Many studies have shown that the increased size of PEGylation leads to a systemic decrease in blood absorption (Caliceti 2003, Kaminskas, Kota et al. 2009). Pegylation may hinder the blood absorption of aptscl56.

In the pharmacokinetic study of PEG40K-aptscl56, absorption takes 60 hours, but elimination takes 35 hours. However, the time required to eliminate most aptamer-PEG is always much longer than absorption (Christopher E. Tucker 1999, Siller-Matula, Merhi et al. 2012). Some studies have shown that the increased size of PEGylation promotes the accumulation of infiltrated tissue through passively enhanced penetration and retention mechanisms (Caliceti 2003). The accumulation of PEG40K-aptscl56 in the permeable tissue may be one of the reasons for the rapid decline in the elimination phase of the pharmacokinetic curve. Further research on this pharmacokinetic phenomenon of PEG40K-aptscl56 will give a clear explanation.

The dosing interval of a multi-dose aptamer can be defined based on its elimination half-life and the dose ratio of the loading dose to the maintenance dose (Birkett 1996, Jambhekar 2012). The elimination half-life of PEG40K-aptscl56 is 66.9 hours. If the dose ratio is 2, the dosing interval is equal to the elimination half-life (T1/2). If the dose ratio is less than 2, the dosing interval should be longer than T1/2. In the pharmacodynamic study of PEG40K-aptscl56, the recommended dose ratio is 1 (the loading dose is equal to the maintenance dose). Therefore, the administration interval of PEG40K-aptscl56 should be longer than 66.9h.

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Claims (10)

1. A method for detecting the presence and/or the expression level of bone sclerostin in a biological sample includes:

   a) Under the condition that a complex can be formed between the aptamer and the sclerostin, the biological sample and the control sample are respectively contacted with the aptamer for the sclerostin;

   b) detect the formation of complexes,

   Wherein the difference in the formation of the complex between the biological sample and the control sample indicates the presence of bone sclerostin and/or the expression level of bone sclerostin in the sample,

   Aptamer

   i) Contains at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity with any of SEQ ID NO: 1-17 Or a nucleotide sequence that is at least about 95% identical, or,

   ii) comprising at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides in any one of SEQ ID NO: 1-17,

   Preferably, the aptamer comprises the sequence of any one of SEQ ID NO: 1-17 or 19-24,

   The aptamer specifically binds to bone sclerostin.
2. The method of claim 1, wherein the aptamer has a K _{d of} less than 100 nM for osteosclerosis, 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.
3. The method of claim 1 or 2, wherein the aptamer for sclerostin is conjugated with a fluorescent dye, chemical substance, polypeptide, enzyme, isotope or label that can be used for detection.
4. Use of an aptamer for osteosclerosin as defined in any one of claims 1 to 3 in the preparation of a kit for detecting the presence and/or the expression level of osteosclerosin in a biological sample .
5. A method for diagnosing a disease related to bone sclerostin in a subject, the method comprising using the aptamer for sclerostin as defined in any one of claims 1 to 3 to detect a person suspected of having a disease related to sclerostin The presence of bone sclerostin and/or the expression level of bone sclerostin in the biological sample of the subject, wherein the presence of bone sclerostin and/or the expression level of bone sclerostin represents the existence and/or state of bone sclerostin-related diseases .
6. A method for diagnosing a disease related to bone sclerostin in a subject by administering an aptamer for sclerostin as defined in any one of claims 1 to 3 to a person suspected of having a disease related to sclerostin The subject is then detected for the accumulation of the aptamer in the subject, thereby diagnosing osteosclerosin-related diseases.
7. The method of claim 6, wherein detecting the accumulation of the aptamer in the subject is achieved by imaging the subject, the aptamer being labeled with an isotope, for example.
8. Use of an aptamer against osteosclerosin as defined in any one of claims 1 to 3 in the preparation of a kit for diagnosing osteosclerosin-related diseases in a subject.
9. The method of any one of claims 5-7 or the use of claim 8, wherein the osteosclerosin-related disease is, for example, a disease in which the expression level of osteosclerosin is abnormal in a subject.
10. A kit for the detection method of any one of claims 1-3 and/or the diagnostic method of any one of claims 5-7, the kit comprising any one of claims 1-3 The defined aptamer for osteosclerosin, a reagent for detecting the presence and/or amount of the aptamer for osteosclerosin, and instructions for use.

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