A bone-forming polypeptide and its application

Abstract

This invention belongs to the field of biomedical materials technology, specifically relating to a bone-forming polypeptide and its applications. Specifically, this invention provides polypeptides or variants thereof for promoting bone formation, bone regeneration, bone repair and / or preventing and / or treating osteoporosis, fusion proteins comprising such polypeptides or variants thereof, pharmaceutical compositions, and the pharmaceutical uses of such polypeptides or variants thereof and fusion proteins.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a bone-forming polypeptide and its applications. Background Technology

[0002] Currently, the only bone-forming drug available in clinical practice is parathyroid hormone-derived peptide (PTH 1-34, teriparatide). Evidence-based clinical data indicate that intermittent use of teriparatide is an effective method for treating osteoporosis. However, its benefits for fracture healing and bone regeneration still require further evaluation through clinical research (Eben GE, Clifford J.R. Emerging insights into the comparative effectiveness of anabolic therapies for osteoporosis. Nat. Rev. Endocrinol. 17, 31-46 (2021)).

[0003] Previous studies have shown that the classical WNT / β-catenin signaling pathway plays a wide role in bone homeostasis, bone development, bone regeneration, and bone metabolism. Activation of the classical WNT pathway can promote bone formation in animal models (Tristan W.F. et al., Development of selective bispecific Wnt mimetics for bone loss and repair. Nat. Commun. 12, 3247 (2021)). Therefore, the classical WNT signaling pathway is also an important target for the development of bone regeneration drugs. Classical WNT agonists such as romosozumab and recombinant WNT3A (rhWNT3A) have been used in the field of promoting bone formation. However, in the process of practical application and clinical translation, it has been found that the use of such classical WNT agonist drugs may lead to an increased risk of tumorigenesis and significant adverse reactions due to the overactivation of classical WNT / β-catenin signaling. These significant risks limit the development and practical application of related drugs.

[0004] Therefore, there is an unmet need in the field for developing new treatments and drugs that can promote bone growth without activating the classic WNT signaling pathway. Summary of the Invention

[0005] Previous research by the inventors has shown that the bone anabolic function of WNT7B (Wnt family member 7B) is independent of β-Catenin and carries no risk of activating the classical WNT pathway (Fanyuan Y. et al., Wnt7b-induced Sox11 functions enhance self-renewal and osteogenic commitment of bone marrow mesenchymal stem cells. Stem Cells. 38, 1020-1033 (2020)). Therefore, this invention aims to demonstrate, through the development of peptides derived from the Thumb and index domains of WNT7B, that WNT7B-derived peptides can exert osteogenic effects in osteoporosis and extreme bone defect models without activating the classical WNT signaling pathway. The therapeutic effects of these derived peptides on osteoporosis and their efficacy in promoting fracture repair and regeneration have been verified in multiple species, including mice and pigs. This effect is closely related to enhanced MSC self-renewal and osteogenic differentiation, and is caused by the activation of Reck / Gpr124 / Ca by WNT7B-derived peptides. 2+ / Nfatc1 signal axis mediation. Therefore, this invention aims to address the shortcomings of current WNT activators in clinical applications and provide novel bone formation-promoting drugs, filling the gap in the development of low-risk bone formation drugs and providing a new strategy for the treatment of bone loss and bone injury diseases such as osteoporosis and fracture healing.

[0006] Isolated polypeptides or their variants

[0007] In one aspect, the present invention provides an isolated polypeptide or a variant thereof, wherein the polypeptide is composed of amino acids 200-217 and 323-336 of the WNT7B protein;

[0008] The variant differs from the polypeptide from which it originates in the substitution (e.g., conserved substitution), deletion, or addition of one or more amino acid residues (including but not limited to 1, 2, 3, 4, 5, or more), while retaining the biological function of the polypeptide from which it originates.

[0009] In this document, the biological functions of the polypeptides or variants thereof of the present invention include, but are not limited to, promoting bone formation, bone regeneration, bone repair and / or treating osteoporosis.

[0010] In some embodiments, the WNT7B protein is human-derived. In some embodiments, the WNT7B protein has the amino acid sequence shown in SEQ ID NO: 1.

[0011] In some embodiments, the isolated polypeptide of the present invention is composed of amino acids 200-217 and 323-336 of the WNT7B protein directly linked together.

[0012] In some embodiments, amino acids 200-217 of the WNT7B protein correspond to the Thumb domain of WNT7B, the sequence of which is shown in SEQ ID NO: 2.

[0013] In some embodiments, amino acids 323-336 of the WNT7B protein correspond to the index domain of WNT7B, the sequence of which is shown in SEQ ID NO: 3.

[0014] In some embodiments, the isolated polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 4.

[0015] In some embodiments, the variants comprise amino acid sequences as shown in SEQ ID NO: 5-7.

[0016] Fusion protein

[0017] In another aspect, the present invention provides a fusion protein comprising the isolated polypeptide (or a variant thereof) of the present invention and another polypeptide.

[0018] In some embodiments, the additional polypeptide is selected from protein tags, targeting moieties, or any combination thereof.

[0019] In this document, protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select appropriate protein tags according to the desired purpose (e.g., purification, detection, or tracing).

[0020] In this document, the term "targeting portion" refers to a domain capable of directing the polypeptide (or a variant thereof) of the present invention to a desired location, which may be a specific tissue, a specific cell, or even a specific intracellular location (e.g., the nucleus, ribosome, endoplasmic reticulum, lysosome, or peroxisome). Those skilled in the art know how to design corresponding targeting domains based on the characteristics of the desired location. In some embodiments, the targeting portion includes a ligand, receptor, or antibody, or a binding domain thereof.

[0021] In some embodiments, the additional polypeptide is optionally linked to the N-terminus or C-terminus of the polypeptide (or a variant thereof) of the present invention via a linker. In some embodiments, the linker is a sequence comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., Gly or Ser).

[0022] Preparation of peptides and fusion proteins

[0023] The polypeptides or their variants or fusion proteins of the present invention are not limited by their manner of production. For example, they can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods, such as solid-phase synthesis on a suitable resin.

[0024] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide or a variant or fusion protein thereof.

[0025] In another aspect, the present invention also provides a vector comprising the isolated nucleic acid molecules as described above. The vector of the present invention can be a cloning vector or an expression vector. In some embodiments, the vector of the present invention is, for example, a plasmid, a phage, a cosmid, etc.

[0026] In another aspect, the present invention also provides host cells comprising the isolated nucleic acid molecules or vectors of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells (e.g., Sf9 cells), plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0027] In another aspect, the present invention also provides a method for preparing the polypeptide or variant or fusion protein of the present invention, comprising culturing the host cell of the present invention under conditions that allow expression of the polypeptide or variant or fusion protein, and recovering the polypeptide or variant or fusion protein from the cultured host cell culture.

[0028] Pharmaceutical Composition

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising the isolated polypeptide (or a variant thereof), fusion protein, isolated nucleic acid molecule, carrier or host cell of the present invention, and pharmaceutically acceptable carrier and / or excipient.

[0030] In some embodiments, the pharmaceutical composition comprises one or more of the isolated polypeptides (or variants thereof) or fusion proteins of the present invention.

[0031] The polypeptides (or variants thereof), fusion proteins, or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, and injections (including injectable solutions and lyophilized powders). In some embodiments, the polypeptides (or variants thereof), fusion proteins, or pharmaceutical compositions of the present invention can be formulated into injectable solutions or lyophilized powders.

[0032] Furthermore, the polypeptides (or variants thereof) or fusion proteins of the present invention may be present in the pharmaceutical composition in unit dose form for ease of administration.

[0033] The polypeptides (or variants thereof), fusion proteins, or pharmaceutical compositions of the present invention may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the polypeptides (or variants thereof), fusion proteins, or pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0034] The polypeptides (or variants thereof), fusion proteins, or pharmaceutical compositions provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., bone-forming agents). Such other pharmaceutically active agents can be administered before, simultaneously with, or after the administration of the polypeptides (or variants thereof), fusion proteins, or pharmaceutical compositions of this invention.

[0035] In some embodiments, the pharmaceutical composition optionally further comprises additional pharmaceutically active agents, such as other drugs that promote bone formation, bone regeneration and / or bone repair, such as bone-forming drugs, such as teriparatide.

[0036] Therapeutic uses

[0037] The inventors of this application have discovered for the first time that by assembling the Thumb and index domains of WNT7B, the resulting WNT7B-derived peptides can exert an osteopromoting effect in osteoporosis and extreme bone defect models without activating the classical WNT signaling pathway.

[0038] Therefore, in another aspect, the present invention also relates to the use of the isolated polypeptides (or variants thereof), fusion proteins, isolated nucleic acid molecules, carriers or host cells of the present invention in the preparation of medicaments for promoting bone formation, bone regeneration, bone repair and / or preventing and / or treating osteoporosis in subjects.

[0039] In some embodiments, the subject may be a mammal, such as a human or a mouse. In some embodiments, the subject is any subject who needs to promote bone formation, bone regeneration, and / or bone repair. In some embodiments, the subject has or is suspected of having osteoporosis, or is at risk of developing the aforementioned disease.

[0040] Terminology Definition

[0041] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0042] As used herein, WNT7B is a member of the WNT gene family, which consists of structure-associated genes encoding secretory signaling proteins. These proteins are involved in tumorigenesis and various developmental processes, including the regulation of cell fate and patterns during embryogenesis. WNT7B is well known to those skilled in the art, and its sequence can be found in various public databases, such as NCBI GENBANK accession number: NM_058238.3.

[0043] As used herein, when referring to the amino acid sequence of the WNT7B protein, the sequence shown in SEQ ID NO: 1 is used for description. For example, the expression "amino acid residues 200-217 of the WNT7B protein" refers to amino acid residues 200-217 of the polypeptide shown in SEQ ID NO: 1. However, those skilled in the art will understand that mutations or variations can be naturally generated or artificially introduced into the amino acid sequence of WNT7B without affecting its biological function. Therefore, in this invention, the term "WNT7B" and similar expressions should include all such sequences, including, for example, the sequence shown in SEQ ID NO: 1 and its natural or artificial variants. Furthermore, when describing a sequence fragment of the WNT7B protein, it includes not only the sequence fragment of SEQ ID NO: 1 but also the corresponding sequence fragment in its natural or artificial variants. For example, the expression "amino acid residues 200-217 of the WNT7B protein" includes amino acid residues 200-217 of SEQ ID NO: 1 and the corresponding fragment in its variants (natural or artificial). According to the present invention, the term "corresponding sequence segment" or "corresponding segment" refers to the segment in the compared sequence that is located at the same position when the sequence is optimally aligned, that is, when the sequence is aligned to obtain the highest percentage of identity.

[0044] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0045] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0046] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0047] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0048] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0049] As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals such as humans. In some embodiments, the subject (e.g., a human) suffers from osteoporosis.

[0050] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19). th (ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, ionic strength enhancers, osmotic pressure maintaining agents, delayed absorption agents, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Delayed absorption agents include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Adjuvants include, but are not limited to, aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired activity of an active ingredient in a drug (e.g., inhibitory activity against PSD-95 ubiquitination), including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0051] As used in this article, the term “treatment” means treating or curing a disease (such as osteoporosis), delaying the onset of one or more symptoms of a disease, and / or slowing the progression of a disease.

[0052] As used herein, the term "effective amount" refers to an amount that is effective in achieving the intended purpose. For example, a therapeutically effective amount can be an amount that is effective or sufficient to treat or cure a disease (e.g., osteoporosis), delay the onset of one or more symptoms of the disease, and / or slow the progression of the disease. Such an effective amount can be readily determined by someone skilled in the art or by a physician and can be related to the intended purpose, the general health condition of the subject, age, sex, weight, the severity of the disease to be treated, complications, method of administration, etc. The determination of such an effective amount is entirely within the capabilities of someone skilled in the art.

[0053] As used herein, the biological functions of the polypeptides or variants thereof of the present invention include, but are not limited to, promoting bone formation, bone regeneration, bone repair and / or preventing and / or treating osteoporosis.

[0054] Beneficial effects

[0055] 1) The WNT7B-derived polypeptide designed in this invention can significantly promote osteogenic differentiation of human mesenchymal stem cells in vitro, and its effect is more significant compared with the existing recombinant WNT3A protein in clinical practice. Moreover, it does not activate the classical WNT pathway. In view of this, it is expected to solve the serious side effects (such as tumorigenesis) that may be caused by the in vivo use of existing WNT agonists, and lay the foundation for the implementation of subsequent in vivo experiments.

[0056] (2) The WNT7B-derived polypeptide designed in this invention can significantly improve two types of osteoporosis through in vivo injection, including mouse estrogen-related (oophorectomy model) osteoporosis and age-related osteoporosis (age-related aging model).

[0057] (3) The WNT7B-derived peptide achieved ideal bone regeneration results in the extreme bone defect model of large mammals—miniature pigs. Sequencing and other molecular biology experiments confirmed that multiple MSC subsets were mobilized to invade and undergo osteogenic differentiation during this process, without activating the classical WNT signaling pathway, indicating that the WNT7B-derived peptide has cross-species therapeutic potential.

[0058] (4) The WNT7B-derived peptide does not activate the classical WNT signaling pathway in vivo or in vitro. Instead, it enhances the self-renewal and osteogenic differentiation of MSCs through the activation of Ca2+ / Nfatc1 mediated by the Reck / Gpr124 signaling body.

[0059] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0060] Figure 1 Development of WNT7B-derived peptides. Figure A shows the predicted structure of the WNT7B protein. The positions of the Thumb and index domains are specially marked with dashed lines. Figure B shows the sequence of a single Thumb and index domain of the human WNT7B protein. Figure C shows the sequences of the derived peptide 1797 and its variants 1798, 1799, and 1800 designed in this invention.

[0061] Figure 2The in vitro osteogenic effect of WNT7B-derived peptides. Figure A shows the quantification results of Alizarin Red staining of human mesenchymal stem cells (hMSCs) treated with different concentrations of four derivative peptides (cultured in osteogenic induction medium (OM) for 28 days); Figure B shows the assessment of key osteogenic gene levels by RT-qPCR.

[0062] Figure 3 The osteogenic effect of the 1797 peptide was further validated in an in vitro simulated bone engineering system. Figure A shows the mineralized nodules of hMSCs induced on the 1797 scaffold using OM medium for 21 days; Figure B shows the distribution of C, O, Ca, and P elements after 21 days.

[0063] Figure 4 In vivo injection of the 1797 peptide significantly alleviated osteoporosis in two models. Figure A shows μCT images of the lumbar spine in a mouse model of estrogen-related osteoporosis mediated by ovariectomy after different treatments; Figure B shows a representative μCT image of the femoral trabeculae in an age-related osteoporosis model.

[0064] Figure 5 Single-cell sequencing results after applying the 1797 peptide scaffold in a miniature pig model of extreme bone defects. Figure A shows the differential cell populations after dimensionality reduction analysis of mesenchymal stem cell populations; Figure B shows the cell numbers of MSC subsets in the control and 1797 groups; Figure C shows gene enrichment analysis (GSEA) data on β-Catenin-dependent classical WNT signaling within MSC lineages in the control and 1797 groups.

[0065] Figure 6 Immunofluorescence staining (activated β-Catenin, ABC) was performed on samples obtained from a mouse model of extreme bone defects. Figure A shows the statistical results of the number of positive cells per unit area; Figure B shows the activation level of the WNT signaling pathway as detected by TOP / FOP dual-luciferase assay; Figure C shows the β-Catenin-dependent characteristic gene level in hMSCs analyzed by RT-qPCR; Figure D is a violin diagram of Nfatc1 expression in MSC lineages in a miniature pig model of extreme bone defects; Figure E summarizes the mechanism by which the 1797 peptide promotes bone function without activating the classical WNT pathway.

[0066] Figure 7 Example 7: Evaluation of the osteopromoting effect of 1797 peptide in aging-related osteoporosis in miniature pigs.

[0067] Figure 8Example 8: Analysis of the osteopromoting effect of 1797 polypeptide scaffold treatment in a miniature pig extreme bone defect model. Figure A shows the effect after transplantation of the 1797 polypeptide scaffold; Figure B shows the relative mineralization of the defect area 6 months after surgery (based on μCT results); Figure C is a representative H&E stained image; Figure D shows the behavioral assessment results of animals after implantation of the control scaffold and the 1797 polypeptide scaffold. Detailed Implementation

[0068] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0069] Example 1: Development of WNT7B-derived peptides

[0070] This invention designs a series of peptides based on the WNT7B Thumb and index domains. The specific experimental procedures are as follows:

[0071] (1) The Thumb and index domains are indispensable for the ligand selectivity signal of WNT7B. The Thumb domain is located near the N-terminus (amino terminus) and has the sequence KCHGVSGSCTTKTCWTT (SEQ ID NO: 2), while the index domain is located near the C-terminus (carboxyl terminus) and has the sequence QCNCKFHWCCFVKC (SEQ ID NO: 3). Figure 1 AB);

[0072] (2) Design peptides based on the WNT7B Thumb and index domains. Figure 1 As shown in C, the involved polypeptide 1 (1797) consists of 31 amino acids, with the specific sequence KCHGVSGSCTTKTCWTTQCNCKFHWCCFVKC (SEQ ID NO: 4); the variant polypeptide 2 (1798) consists of 28 amino acids, with the sequence KCHGVSGSCGPGGDQCNCKFHWCCFVKC (SEQ ID NO: 5); the variant polypeptide 3 (1799) consists of 19 amino acids, with the sequence GPGGDQCNCKFHWCCFVKC (SEQ ID NO: 6); and the variant polypeptide 4 (1800) consists of 14 amino acids, with the sequence GPGGDKCHGVSGSC (SEQ ID NO: 7).

[0073] All peptides in this invention are synthesized by chemical methods outsourced to a company.

[0074] Example 2: In vitro osteogenic effect of WNT7B-derived peptides

[0075] The in vitro osteogenic effects of the peptides and their variants designed in Example 1 were evaluated. The results are as follows: Figure 2 As shown, all of these peptides exhibited good in vitro osteopromoting effects, with 1797 showing the best results. The specific experimental procedure is as follows:

[0076] (1) Human mesenchymal stem cells (hMSCs) were cultured in osteogenic induction medium (OM) for 28 days;

[0077] (2) Remove the culture medium, wash the cells twice with 1xPBS (without calcium and magnesium ions), and then fix the cells at room temperature for 15 min;

[0078] (3) Remove the fixative, wash the cells twice with PBS, then cover the sample with alizarin red stain and incubate at 37°C in the dark for 60 minutes.

[0079] (4) After washing away excess staining solution with PBS, quantitatively analyze alizarin red. For example... Figure 2 As shown, 1797 had the best osteogenic effect in cultured hMSCs, with a significant difference compared to the control group. Although other variants also had some osteogenic effect, the effect was not as significant as that of 1797.

[0080] (5) RNA was extracted, cDNA was synthesized and PCR amplified from hMSCs cultured under the same conditions. The levels of key osteogenic genes, including ALPL, COL1A1, RUX2 and SP7, were detected by RT-qPCR, with recombinant WNT3A protein as a positive control. Compared with recombinant WNT3A protein, the 1797 peptide designed in this invention has a stronger osteogenic induction effect on hMSCs.

[0081] Example 3: Evaluation of the osteopromoting effect of 1797 peptide in human cells

[0082] The promoting effect of 1797 peptide on osteogenic differentiation of hMSCs was observed in an in vitro simulated bone engineering system. The specific experimental procedure is as follows:

[0083] (1) Constructing an in vitro simulated skeletal engineering system:

[0084] a. Pure hyaluronic acid (HA) scaffolds were immersed in graphene oxide (GO) alcohol solution for 30 minutes and dried at 60°C to obtain HA / GO scaffolds;

[0085] b. HA / GO was further immersed in a composite solution (ethylenediamine (EDA): dimethylformamide (DMF) ratio of 1:4) at 40°C for 12 hours to graft the amino group -NH2 onto the carboxyl group -COOH of GO to obtain HA / GO-NH2;

[0086] c. Immerse the HA / GO-NH2 scaffold in a 2wt% sodium ascorbate (NaVC) aqueous solution at 60°C for 8 hours to reduce additional oxidized functional groups on GO-HN2;

[0087] d. HA / rGO-NH2 was immersed in 1797 polypeptide aqueous solution (2 mg / mL) at 15 °C for 48 hours to prepare HA / rGO-1797 scaffold;

[0088] (2) The modified HA / rGO-1797 scaffold was placed in a well plate, hMSCs were seeded onto the scaffold, and the cells were cultured in OM medium for 21 days.

[0089] (3) After 21 days, the deposition of cell mineralization nodules on the 1797 scaffold and the control scaffold was observed by transmission electron microscopy (TEM). Figure 3 The results of A indicate that the number of mineralized nodules on the 1797 scaffold was significantly increased compared with the control scaffold, suggesting that 1797 promoted the mineralization of hMSCs.

[0090] (4) Element mapping showed that the contents of elements Ca and P on the 1797 scaffold were significantly higher than those on the control scaffold. Figure 3 B) indicates that the 1797 peptide can lead to a significant enhancement in osteogenic and mineralization of hMSCs.

[0091] Example 4: Evaluation of the osteoproliferative effect of 1797 peptide in a mouse osteoporosis model

[0092] like Figure 4 As shown, the 1797 peptide designed in this invention exerted a bone-promoting effect in both mouse osteoporosis models, effectively alleviating disease progression. The specific experimental procedure is as follows:

[0093] (1) Establishment of an osteoporosis model mediated by oophorectomy (OVX):

[0094] a. After anesthetizing the mouse with 3% sodium pentobarbital intraperitoneally, the mouse was placed in a prone position, and the hair on the back was removed about 1 cm to the left and right of the highest point of the mouse's spine.

[0095] b. Under aseptic conditions, the skin and muscle in the hair removal area were bluntly dissected, and white adipose tissue could be seen through the parietal peritoneum to locate the mouse ovary.

[0096] c. Use No. 1 thread to perform a circular ligation at the junction of the ovary and fallopian tube, and remove the ovary with surgical scissors;

[0097] d. Push the adipose tissue back into the abdominal cavity, suture the tunica albuginea and muscle layer together, and then suture the skin;

[0098] e. The sham group received the exact same procedures as the experimental group except for the removal of the ovaries;

[0099] f. Two months after the surgery, the experimental mice were euthanized and further analysis was performed.

[0100] (2) Establishment of an age-related osteoporosis model: Mice survived in an SPF environment for 24 months. During this period, the mice were housed in a well-equipped facility with a temperature of 20-25°C and a humidity of 30%-70%, and were subjected to the same dark / light cycle (12:12). After 24 months, the mice were euthanized and the next experimental procedure was performed.

[0101] (3) Figure 4 The results showed that in vivo injection of 1797 peptide significantly alleviated estrogen deficiency-related (OVX model) and age-related osteoporosis in mice. Specifically, μCT images showed increased trabecular bone mass in the 1797 treatment group compared to the control group, ultimately eliminating the osteoporotic phenotype of the OVX group. Figure 4 A);

[0102] (4) In an age-related osteoporosis model, injection of 1797 peptide significantly increased trabecular bone mass in aged mice and effectively alleviated osteoporosis. Figure 4 B).

[0103] Example 5: Evaluation of the role of the 1797 polypeptide scaffold in a miniature pig model of extreme bone defects

[0104] In a large mammalian model of critical bone defects, a 1797 peptide scaffold was used. Single-cell sequencing verified that the mechanism promoting bone formation and repair was a response of the MSC population. The specific experimental procedure is as follows:

[0105] (1) A model of extreme bone defects was constructed using miniature pigs;

[0106] (2) The engineered 1797 scaffold was transplanted into the bone defect cavity. One month after transplantation, the scaffold was retrieved and digested. Single-cell RNA sequencing (scRNA-seq) was performed on the digested cells.

[0107] (3) From Figure 5 It can be seen that, in the mesenchymal components of the 1797 polypeptide scaffold-treated group, the cell subpopulations that showed significant differences compared to the control group were skeletal stem cells (SSC), mesenchymal stem cells (MSC), and mesenchymal progenitor cells (MPC). The control group had almost no MSC, SSC, and MPC subpopulations, but the number of these subpopulations was significantly increased in the 1797 group. Figure 5 A);

[0108] (4) The osteoblast (OB) subset in the 1797 polypeptide scaffold group also showed an increase, indicating enhanced bone formation. Figure 5 B);

[0109] (5) Gene set enrichment analysis (GSEA) of the mesenchymal fraction showed that, compared with the control group, the 1797 polypeptide scaffold group did not exhibit typical WNT activation, indicating that the in vivo effect of 1797 on MSCs is β-Catenin-independent. Figure 5 C).

[0110] Example 6: The 1797 peptide does not activate the classical WNT signaling pathway.

[0111] Further confirmation indicates that the 1797 peptide does not activate the classical WNT signaling pathway in vivo or in vitro. Instead, it enhances MSC self-renewal and osteogenic differentiation through Ca2+ / Nfatc1 activation mediated by the Reck / Gpr124 signaling neurotransmitter. The specific experimental procedure is as follows:

[0112] (1) Construct a mouse model of extreme bone defects;

[0113] (2) Decalcify the sample using 10%-12% EDTA. After the sample softens, dehydrate it overnight at 4°C using a 30% sucrose solution.

[0114] (3) The OCT sample was pre-soaked at 4℃ for 3 hours, the sample angle was adjusted, and it was frozen and embedded at -20℃. After the sample was frozen, it was sectioned using a cryostat to a thickness of 6μm;

[0115] (4) Immunofluorescence staining: Frozen tissue sections were immersed in PBS for 15 minutes. 0.5% Triton was permeabilized for 10 minutes at room temperature, and BSA powder was dissolved in 0.5% Triton to obtain a Triton solution containing 3% BSA for sample blocking. The primary antibody (activated β-Catenin, ABC) was diluted to the recommended concentration according to the manufacturer's instructions and incubated overnight at 4°C. The primary antibody was washed away with PBS, and the secondary antibody was diluted with DAPI solution to the recommended concentration according to the manufacturer's instructions and incubated at room temperature for 2 hours. The secondary antibody was washed away with PBS three times, 10 minutes each time. Finally, the sections were mounted with anti-fluorescence attenuation mounting medium, and images were acquired using laser confocal microscopy and statistically analyzed using ImageJ.

[0116] (5) Count the number of cells expressing ABC per unit area. Figure 6 A) The results showed that the 1797 peptide did not activate β-Catenin in mice;

[0117] (6) The activation status of the WNT pathway was detected by TOP / FOP dual-luciferase assay. Figure 6B shows that, compared with recombinant WNT3A, treatment with the 1797 peptide did not activate the classical WNT signaling pathway in either human or mouse mesenchymal cell lines.

[0118] (7) Further verification by RT-qPCR showed that, compared with recombinant WNT3A, 1797 treatment did not induce β-Catenin-dependent gene expression in hMSCs. Figure 6 C);

[0119] (8) Further analysis of single-cell sequencing results of cells grown after implantation of the 1797 polypeptide scaffold in the miniature pig extreme bone defect in Example 5 revealed that the Nfatc1 positive cell population was significantly upregulated in the MSC lineage. Figure 6 D);

[0120] (9) It has been previously reported that Ca2+ / Nfatc1 signaling controls the self-renewal and osteogenic differentiation of Nfatc1+MSCs. Therefore, the 1797 peptide can induce the self-renewal and osteogenic differentiation of Nfatc1+MSCs through the activation of Ca2+ / Nfatc1 mediated by the Reck / Gpr124 signaling body, thereby exerting an in vivo and in vitro osteogenic effect. Figure 6 E).

[0121] Example 7: Evaluation of the osteopromoting effect of 1797 peptide in a miniature pig model of aging-related osteoporosis

[0122] This embodiment analyzes the effects of WNT7B-derived peptide 1797 on disease progression and bone promotion in a miniature pig model of aging-related osteoporosis. The specific experimental procedure is as follows:

[0123] (1) Throughout the experiment, the miniature pigs were placed under a constant light / dark schedule of 12 hours / 12 hours, with a constant temperature of 20°C and a constant relative humidity of 50%.

[0124] (2) Miniature pigs can drink water freely in addition to receiving pelleted commercial food twice a day;

[0125] (3) Minimize animal suffering and reduce the number of animals used during the experiment;

[0126] (4) All animals were monitored by veterinarians during the experiment;

[0127] (5) Starting from the age of 6, miniature pigs were injected intraperitoneally with 1797 polypeptide daily for three consecutive months. After three months, the animals were euthanized and femoral tissue was obtained for CT analysis and reconstruction.

[0128] (5) Figure 7μCT images showed that, compared with the control group, in vivo application of 1797 peptide significantly increased the number, thickness and bone mass of trabeculae in aged pigs, while reducing trabecular separation at the distal femoral metaphysis; 1797 continuously reduced the porosity of cortical bone in aged pigs and increased the bone mineral density of cortical bone.

[0129] This embodiment demonstrates that applying WNT7B-derived peptide 1797 to age-related osteoporosis in miniature pigs can effectively alleviate disease symptoms and increase bone mass.

[0130] Example 8: Effects of 1797 peptide on bone regeneration and animal recovery in a miniature pig model of extreme bone defect.

[0131] This embodiment applies WNT7B-derived polypeptide 1797 in a miniature pig model of extreme bone defect to observe the bone regeneration effect and animal recovery. The specific experimental procedure is as follows:

[0132] (1) Constructing a critical femoral fracture model using miniature pigs:

[0133] a. To ensure acclimatization, the miniature pigs were quarantined for five to ten days before the experiment began;

[0134] b. In accordance with ethical standards, miniature pigs were euthanized within 6 months of stent implantation. The femoral condyle was dissected, and the skin and muscle were incised with a scalpel and scissors. The entire femoral condyle was removed using a circular saw;

[0135] c. Fix the tissue specimens at room temperature on a 100 ml shaking table with 4% neutral buffered formalin for five days;

[0136] d. General anesthesia was administered during the surgery, and prophylactic amoxicillin (20 mg / kg; Centrafarm) was given intravenously. Meloxicam (15 mg / mL; Metacam; Boehringer) was administered preoperatively as an analgesic; in addition, to reduce the risk of infection, antibiotics (streptomycin 0.5 g / day) were administered one hour before anesthesia induction and for two days after anesthesia induction.

[0137] e. Disinfect the surgical site by wiping it with 4% chlorhexidine gluconate surgical scrub, and then make a longitudinal incision on the inner side of the right knee.

[0138] f. Incise the subcutaneous tissue and periosteum to expose the surface of the femoral condyle. Use a bone trephine to prepare a cavity at 2000 rpm under saline irrigation (0.9% NaCl). The exposed bone cavity for each animal was 10 mm in diameter and 6 mm in depth.

[0139] (2) The 1797 polypeptide scaffold was transplanted into the above-mentioned femoral cavity, and micro-CT analysis was performed 6 months after transplantation. Figure 8Data from μCT scans showed that the 1797 peptide scaffold group possessed abundant new bone, while the control group showed no significant regeneration of bone tissue. Histological staining results indicated that 6 months post-transplantation, the control group exhibited significant rejection, filled with unmineralized fibrous tissue, while the defect area in the 1797 peptide scaffold group was occupied by newly formed bone, and the scaffold and host bone had integrated into a single unit. Extensive new bone regeneration also occurred within the scaffold pores, indicating that stem cells proliferated and differentiated osteogenically in the microenvironment present with 1797 peptide. Functional activity tests showed that 1797 peptide scaffold treatment restored almost normal activity in miniature pigs with bone defects, while the control group remained poorly active. This example demonstrates that the application of 1797 peptide in a miniature pig model of critical bone defects can effectively promote new bone formation, restore normal animal activity, and exhibits good biocompatibility.

[0140] It should be noted that while the preferred embodiments of the present invention are provided in this specification, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. An isolated polypeptide, characterized in that, The isolated polypeptide consists of an amino acid sequence as set forth in SEQ ID NO:

4.

2. A fusion protein, characterized in that, The fusion protein is linked to N- or C-terminus of the isolated polypeptide according to claim 1; The additional polypeptide is selected from a protein tag selected from His, Flag, GST, MBP, HA, Myc, GFP or biotin.

3. An isolated nucleic acid molecule, comprising, The nucleic acid molecule encodes the isolated polypeptide according to claim 1 or the fusion protein according to claim 2.

4. A vector, characterized by, comprising the isolated nucleic acid molecule according to claim 3.

5. A host cell, characterized in that, comprising the isolated nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. A pharmaceutical composition, characterized by, comprising the isolated polypeptide according to claim 1, the fusion protein according to claim 2, the isolated nucleic acid molecule according to claim 3, the vector according to claim 4 or the host cell according to claim 5, and a pharmaceutically acceptable carrier and / or excipient.

7. The pharmaceutical composition of claim 6, wherein, The pharmaceutical composition further comprises an additional pharmaceutically active agent.

8. The pharmaceutical composition of claim 7, wherein, The additional pharmaceutically active agent is another drug having the effect of promoting bone formation, bone regeneration and / or bone repair.

9. Use of the isolated polypeptide according to claim 1, the fusion protein according to claim 2, the isolated nucleic acid molecule according to claim 3, the vector according to claim 4, the host cell according to claim 5 or the pharmaceutical composition according to any one of claims 6 to 8 for the manufacture of a medicament, characterized in that, The drug is used for promoting bone formation, bone regeneration, bone repair.

10. Use according to claim 9, characterized in that, The drug is used for preventing and / or treating osteoporosis. The drug is used for preventing and / or treating osteoporosis.

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