Bone formation promoting polypeptide and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a bone formation promoting polypeptide and application thereof. Specifically, the invention provides a polypeptide or a variant thereof for promoting bone formation, bone regeneration, bone repair and / or preventing and / or treating osteoporosis, a fusion protein containing the polypeptide or the variant thereof, a pharmaceutical composition and medical application of the polypeptide or the variant thereof and the fusion protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a polypeptide promoting bone formation and application thereof. Background Art

[0002] Currently, the only bone-forming drug available in clinical practice is parathyroid hormone-derived peptide (PTH 1-34, teriparatide). Evidence-based clinical data show that intermittent administration of teriparatide is an effective method for treating osteoporosis. However, its benefits on fracture healing and bone regeneration still need more clinical studies to evaluate (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 canonical WNT / β-catenin signaling pathway plays a wide range of roles in bone homeostasis, bone development, bone regeneration, and bone metabolism. Activating the canonical 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 canonical WNT signaling pathway is also an important target for the development of bone regeneration drugs. Canonical 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 transformation, it was found that the use of such canonical WNT agonist drugs may lead to tumor risks and major adverse reactions due to excessive activation of canonical WNT / β-Catenin signals. These non-negligible risks limit the development and practical application of related drugs.

[0004] Therefore, there is an unmet need in the art to develop new therapeutic options and drugs that exert osteogenic effects without activating the canonical WNT signaling pathway. Summary of the invention

[0005] Previous studies by the inventors have shown that the bone anabolic effect of WNT7B (Wnt family member 7B) exists independently of β-Catenin, and there is no risk of activating the canonical WNT pathway (Fanyuan Y. et al., Wnt7b-inducedSox11functions enhance self-renewal and osteogenic commitment of bone marrowmesenchymal stem cells. Stem Cells. 38, 1020-1033 (2020)). Therefore, the present invention intends to develop WNT7B Thumb and index domain-derived polypeptides to prove that WNT7B-derived polypeptides can play a role in promoting osteoporosis and extreme bone defect models by not activating the canonical WNT signaling pathway, and verify the therapeutic effect of the derived polypeptide on osteoporosis and the efficacy of promoting fracture repair and regeneration in multiple species such as mice and pigs. This effect is closely related to the self-renewal and enhanced osteogenic differentiation of MSCs, and the WNT7B-derived polypeptide activates Reck / Gpr124 / Ca 2+ / Nfatc1 signaling axis. Therefore, the present invention intends to solve the drawbacks of current WNT activators in clinical application and provide a new drug for promoting bone formation, 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 in the new generation.

[0006] Isolated polypeptide or variant thereof

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

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

[0009] 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 treating osteoporosis.

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

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

[0012] In certain 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 certain 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 certain embodiments, the isolated polypeptide comprises the amino acid sequence shown in SEQ ID NO:4.

[0015] In certain embodiments, the variant comprises the amino acid sequence 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 of the present invention (or a variant thereof) and another polypeptide.

[0018] In certain embodiments, the additional polypeptide is selected from a protein tag, a targeting moiety, or any combination thereof.

[0019] Herein, protein tags are well known in the art, examples of which 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 a suitable protein tag according to the desired purpose (e.g., purification, detection or tracing).

[0020] In this article, the term "targeting moiety" refers to a domain that can direct a polypeptide of the present invention (or its variant) to a desired location, which can be a specific tissue, a specific cell, or even a specific intracellular location (e.g., a nucleus, a ribosome, an endoplasmic reticulum, a lysosome, or a peroxisome). It is known to those skilled in the art how to design a corresponding targeting domain by the characteristics of the desired location. In certain embodiments, the targeting moiety includes a ligand, a receptor, or an antibody or its binding domain.

[0021] In certain embodiments, the additional polypeptide is optionally connected to the N-terminus or C-terminus of a polypeptide of the present invention (or its variant) via a linker. In certain 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 polypeptide or variant or fusion protein of the present invention is not limited by the way it is produced. For example, it 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 of the present invention or a variant or fusion protein thereof.

[0025] On the other hand, the present invention also provides a kind of vector, it comprises the nucleic acid molecule of separation as above.The vector of the present invention can be a cloning vector, also can be an expression vector.In certain embodiments, the vector of the present invention is for example a plasmid, a cosmid, a phage, a cosmid or the like.

[0026] On the other hand, 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 (such as Sf9 cells), plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0027] On the other hand, the present invention also provides a method for preparing the polypeptide or its variant or fusion protein of the present invention, which comprises culturing the host cell of the present invention under conditions allowing expression of the polypeptide or its variant or fusion protein, and recovering the polypeptide or its 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 variant thereof), fusion protein, isolated nucleic acid molecule, vector or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

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

[0031] The polypeptide (or its variant), fusion protein or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the polypeptide (or its variant), fusion protein or pharmaceutical composition of the present invention can be formulated into injection solutions or lyophilized powders.

[0032] In addition, the polypeptide (or variant thereof) or fusion protein of the present invention may be present in a pharmaceutical composition in a unit dosage form for ease of administration.

[0033] Polypeptide (or its variant), fusion protein or pharmaceutical composition of the present invention can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to the intended purpose. In a preferred embodiment, polypeptide (or its variant), fusion protein or pharmaceutical composition of the present invention is given by intravenous infusion or injection.

[0034] The polypeptide (or its variant), fusion protein or pharmaceutical composition provided by the present invention can be used alone or in combination, or can be used in combination with another pharmaceutically active agent (e.g., a drug that promotes bone formation). Such another pharmaceutically active agent can be used before, simultaneously or after the polypeptide (or its variant), fusion protein or pharmaceutical composition of the present invention is used.

[0035] In certain embodiments, the pharmaceutical composition optionally further comprises another pharmaceutically active agent, such as other drugs having the effect of promoting bone formation, bone regeneration and / or bone repair, such as pro-osteoblastic drugs, such as teriparatide.

[0036] Therapeutic Uses

[0037] The inventors of the present application discovered for the first time that by assembling the thumb and index domains of WNT7B, the obtained WNT7B-derived polypeptide can play a pro-osteoblastic role in osteoporosis and extreme bone defect models by not activating the classical WNT signaling pathway.

[0038] Therefore, on the other hand, the present invention also relates to the use of the isolated polypeptide (or its variant), fusion protein, isolated nucleic acid molecule, vector or host cell of the present invention in the preparation of a medicament for promoting bone formation, bone regeneration, bone repair and / or preventing and / or treating osteoporosis in a subject.

[0039] In certain embodiments, the subject can be a mammal, such as a human or a mouse. In certain embodiments, the subject is any subject that needs to promote bone formation, bone regeneration and / or bone repair. In certain embodiments, the subject suffers from or is suspected of suffering from osteoporosis, or has the risk of suffering from the above-mentioned disease.

[0040] Definition of terms

[0041] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0042] As used herein, WNT7B is a member of the WNT gene family, which is composed of structurally related genes encoding secretory signal proteins. These proteins are related to tumorigenesis and various developmental processes, including the regulation of cell fate and pattern 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 database accession number: NM_058238.3.

[0043] As used herein, when referring to the amino acid sequence of the WNT7B protein, it is described using the sequence shown in SEQ ID NO: 1. For example, the expression "amino acid residues 200-217 of the WNT7B protein" refers to the amino acid residues 200-217 of the polypeptide shown in SEQ ID NO: 1. However, it is understood by those skilled in the art that mutations or variations may occur naturally or be artificially introduced into the amino acid sequence of WNT7B without affecting its biological function. Therefore, in the present invention, the term "WNT7B" and similar expressions shall 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 a sequence fragment of SEQ ID NO: 1, but also a corresponding sequence fragment in its natural or artificial variant. 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 corresponding fragments in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragments" or "corresponding fragments" refers to fragments located at equivalent positions in the compared sequences when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity.

[0044] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0045] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also 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 change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by 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 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 bonds or hydrogen bonds, etc.). 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., 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 compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present 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 certain embodiments, the subject (eg, human) suffers from osteoporosis.

[0050] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th Ed.Pennsylvania:MackPublishing Company,1995), and include but are not limited to: pH regulators, surfactants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Agents for maintaining osmotic pressure include but are not limited to sugars, NaCl and the like. Agents for delaying absorption 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), etc. 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 agents and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug (e.g., inhibitory activity on PSD-95 ubiquitination), including but not limited to sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or their degradation products (e.g., lactalbumin hydrolysate), etc.

[0051] As used herein, the term "treating" refers to treating or curing a disease (eg, osteoporosis), delaying the onset of one or more symptoms of a disease, and / or delaying the progression of a disease.

[0052] As used herein, the term "effective amount" refers to an amount that can effectively achieve 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 a disease, and / or delay the development of a disease. Such an effective amount can be easily determined by a person skilled in the art or a doctor, and can be related to the intended purpose, the general health of the subject, age, sex, weight, severity of the disease to be treated, complications, mode of administration, etc. The determination of such an effective amount is fully within the capabilities of those skilled in the art.

[0053] As used herein, the biological functions of the polypeptides of the present invention or variants thereof 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 the present invention can significantly promote the osteogenic differentiation of human mesenchymal stem cells in vitro, and the effect is more significant than the existing recombinant WNT3A protein in clinical practice, and does not activate the canonical WNT pathway. In view of this, it is expected to solve the serious side effects (tumor occurrence, etc.) that may be caused by the use of existing WNT agonists in vivo, laying the foundation for the implementation of subsequent in vivo experiments;

[0056] (2) The WNT7B-derived polypeptide designed by the present invention can significantly improve two types of osteoporosis through in vivo injection, including estrogen-related (ovarian removal model) osteoporosis and aging-related osteoporosis (aging-related aging model) in mice;

[0057] (3) WNT7B-derived peptides were used in a large mammal, miniature pig, to achieve ideal bone regeneration results in an extreme bone defect model. Sequencing and other molecular biology experiments confirmed that during this process, multiple MSC subsets were mobilized to invade and undergo osteogenic differentiation without activating the canonical WNT signaling pathway, indicating that WNT7B-derived peptides have cross-species therapeutic potential;

[0058] (4) WNT7B-derived peptides do not activate the canonical WNT signaling pathway in vivo or in vitro, but instead enhance the self-renewal and osteogenic differentiation of MSCs through the activation of Ca2+ / Nfatc1 mediated by the Reck / Gpr124 signalosome.

[0059] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 :Development of WNT7B derived polypeptides. Figure A is a predicted structure of WNT7B protein. The positions of thumb and index domains are specially marked with dotted lines; Figure B is the sequence of a single thumb and index domain of human WNT7B protein; Figure C is the sequence of the derived peptide 1797 and its variants 1798, 1799, 1800 designed by the present invention.

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

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

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

[0064] Figure 5 :Single-cell sequencing results after applying 1797 peptide scaffold in miniature pig extreme bone defect model. Figure A shows the different cell populations that appeared after dimensionality reduction analysis of mesenchymal stem cell populations; Figure B shows the number of cells in the control group and 1797 group MSC subpopulations; Figure C shows the gene enrichment analysis (GSEA) data of β-Catenin-dependent canonical WNT signaling within the MSC lineage in the control group and 1797 group.

[0065] Figure 6 :Samples were obtained from the mouse extreme bone defect model for immunofluorescence staining (activated β-Catenin, ABC). 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 detected by TOP / FOP dual luciferase; Figure C shows the level of β-Catenin-dependent characteristic genes in hMSCs analyzed by RT-qPCR. Figure D is a violin plot of Nfatc1 expression in MSC lineage cell populations in the miniature pig extreme bone defect model; Figure E summarizes the relevant mechanisms of 1797 polypeptide in promoting osteogenic function without activating the classical WNT pathway.

[0066] Figure 7 Example 7 Evaluation of the osteogenic effect of 1797 polypeptide in small and medium-sized pigs with aging-related osteoporosis.

[0067] Figure 8Example 8 Analysis of the osteogenic effect of the 1797 polypeptide scaffold in the small and medium-sized pig extreme bone defect model. Figure A shows the effect of the 1797 polypeptide scaffold after transplantation; Figure B shows the relative mineralization degree of the defect area 6 months after surgery (based on μCT results); Figure C is a representative image of H&E staining; Figure D is the animal behavior evaluation results after implantation of the control scaffold and the 1797 polypeptide scaffold. DETAILED DESCRIPTION

[0068] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0070] The present invention designs a series of polypeptides based on the WNT7B thumb and index domains, and the specific experimental process is as follows:

[0071] (1) The thumb and index domains are essential for the ligand-selective signal of WNT7B. The thumb domain is close to the N-terminus (amino terminus) and has the sequence KCHGVSGSCTTKTCWTT (SEQ ID NO: 2), and the index domain is close to 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 polypeptide 1 (1797) involved is composed of 31 amino acids, and the specific sequence is KCHGVSGSCTTKTCWTTQCNCKFHWCCFVKC (SEQ ID NO: 4); the variant polypeptide 2 (1798) of polypeptide 1 is composed of 28 amino acids, and the sequence is KCHGVSGSCGPGGDQCNCKFHWCCFVKC (SEQ ID NO: 5); the variant polypeptide 3 (1799) is composed of 19 amino acids, and the sequence is GPGGDQCNCKFHWCCFVKC (SEQID NO: 6); the variant polypeptide 4 (1800) is composed of 14 amino acids, and the sequence is GPGGDKCHGVSGSC (SEQ ID NO: 7).

[0073] All polypeptides of the present invention are outsourced to companies for synthesis through chemical methods.

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

[0075] The polypeptides and variants designed in Example 1 were used to evaluate the osteogenic effect in vitro. Figure 2 As shown, this series of peptides all have good in vitro osteogenic effects, among which 1797 has the best effect. The specific experimental process is as follows:

[0076] (1) Human mesenchymal stem cells (hMSCs) were cultured in osteogenic 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 minutes;

[0078] (3) Remove the fixative, wash the cells twice again with PBS, then cover the samples with Alizarin Red staining and incubate at 37°C in the dark for 60 min;

[0079] (4) After washing away the excess dye with PBS, quantitative analysis of Alizarin Red was performed. Figure 2 As shown, 1797 has the best osteogenic effect in hMSCs cultured in vitro, with a significant difference compared with the control group. Although other variants also have a certain osteogenic effect, the significance is lower than 1797.

[0080] (5) hMSCs cultured under the same conditions as above were subjected to RNA extraction, cDNA synthesis and PCR amplification, and the levels of key osteogenic genes, including ALPL, COL1A1, RUX2 and SP7, were detected by RT-qPCR, and recombinant WNT3A protein was used as a positive control; compared with the recombinant WNT3A protein, the 1797 polypeptide designed by the present invention had a stronger osteogenic induction effect on hMSCs.

[0081] Example 3: Evaluation of the osteogenic effect of 1797 polypeptide in human cells

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

[0083] (1) Construction of an in vitro simulated bone engineering system:

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

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

[0086] c. The HA / GO-NH2 scaffold was immersed in a 2 wt % sodium ascorbate (NaVC) aqueous solution at 60 ° C for 8 h to reduce the 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 scaffolds were placed in a well plate, hMSCs were seeded on the scaffolds, and the cells were cultured in OM medium for 21 days;

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

[0090] (4) Element mapping showed that the content of Ca and P in the 1797 scaffold was significantly higher than that in the control scaffold ( Figure 3 B), indicating that 1797 peptide can significantly enhance the bio-osteogenesis and mineralization of hMSCs.

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

[0092] like Figure 4 As shown, the 1797 polypeptide designed by the present invention plays a role in promoting osteoblastogenesis in both mouse osteoporosis models and effectively alleviates the disease process. The specific experimental process is as follows:

[0093] (1) Establishment of Ovariectomy (OVX)-mediated Osteoporosis Model:

[0094] a. The mice were anesthetized with 3% sodium pentobarbital and placed in a prone position. The hair on the back was removed at a distance of about 1 cm from the highest point of the spine on the back of the mice.

[0095] b. Bluntly separate the skin and muscles in the depilated area under sterile conditions, and locate the mouse ovaries by visualizing white adipose tissue through the parietal peritoneum;

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

[0097] d. Push the fat tissue back into the abdominal cavity, suture the covering membrane and muscle layer together, and then suture the skin;

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

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

[0100] (2) Establishment of aging-related osteoporosis model: The mice were kept 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, a humidity of 30%-70%, and a same dark / light cycle (12:12). After 24 months, the mice were euthanized and the next experimental operation was performed.

[0101] (3) Figure 4 The results showed that in vivo injection of 1797 peptide significantly alleviated osteoporosis associated with estrogen deficiency (OVX model) and aging in mice. Specifically, the μCT images showed that the trabecular mass of the 1797 treated mice increased compared with the control group, and finally eliminated the osteoporotic phenotype of the OVX group ( Figure 4 A);

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

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

[0104] The 1797 polypeptide scaffold was used in a large mammalian critical bone defect model, and single-cell sequencing was used to verify that the mechanism of promoting bone formation and bone repair was the response of the MSCs population. The specific experimental process is as follows:

[0105] (1) Use miniature pigs to construct an extreme bone defect model;

[0106] (2) The engineered 1797 scaffolds were transplanted into the bone defect cavity, and the scaffolds were harvested and digested one month after transplantation, and 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 component of the 1797 polypeptide scaffold-treated group, the cell subpopulations with significant differences compared with the control group were skeletal stem cells (SSC), mesenchymal stem cells (MSC), and mesenchymal progenitor cells (MPC). MSC, SSC, and MPC subpopulations were almost absent in the control group, but the number of these subpopulations increased significantly in the 1797 group ( Figure 5 A);

[0108] (4) The osteoblast (OB) subpopulation in the 1797 polypeptide scaffold group also increased, indicating enhanced bone formation ( Figure 5 B);

[0109] (5) Gene set enrichment analysis (GSEA) was performed on the mesenchymal part. Compared with the control group, the 1797 polypeptide scaffold group had no typical WNT activation, indicating that the in vivo effect of 1797 on MSCs is independent of β-Catenin ( Figure 5 C).

[0110] Example 6: 1797 polypeptide does not activate the canonical WNT signaling pathway

[0111] It was further confirmed that the 1797 polypeptide did not activate the canonical WNT signaling pathway in vivo or in vitro, but instead enhanced the self-renewal and osteogenic differentiation of MSCs through the activation of Ca2+ / Nfatc1 mediated by the Reck / Gpr124 signalosome. The specific experimental process is as follows:

[0112] (1) Establishment of a mouse extreme bone defect model;

[0113] (2) Decalcify the sample with 10%-12% EDTA. After the sample becomes soft, dehydrate it with 30% sucrose solution at 4°C overnight;

[0114] (3) Soak the sample in OCT at 4°C for 3 hours, adjust the sample angle, and freeze-embed it at -20°C. After the sample is frozen, use a cryostat to slice it to a thickness of 6 μm;

[0115] (4) Immunofluorescence staining: Soak the frozen tissue sections in PBS for 15 minutes. Permeabilize the membrane with 0.5% Triton at room temperature for 10 minutes, dissolve the BSA powder with 0.5% Triton, and obtain a Triton solution containing 3% BSA for sample blocking. Dilute the primary antibody (activated β-Catenin, ABC) according to the concentration recommended in the instructions and incubate overnight at 4°C. Wash off the primary antibody with PBS, dilute the secondary antibody and DAPI solution according to the concentration recommended in the instructions, and incubate at room temperature for 2 hours. Wash off the secondary antibody with PBS, and wash three times for 10 minutes each time. Finally, use anti-fluorescence attenuation mounting medium to seal the sections, acquire images with laser confocal microscope, and perform statistical analysis using imageJ;

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

[0117] (6) Detection of WNT pathway activation by TOP / FOP dual luciferase. Figure 6B shows that compared with recombinant WNT3A, 1797 peptide treatment does not activate the canonical WNT signaling pathway in either human or mouse mesenchymal cell lines;

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

[0119] (8) The results of single-cell sequencing of cells grown after implantation of the 1797 polypeptide scaffold in the small-sized pig extreme bone defect in Example 5 were further analyzed, and it was found 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, 1797 peptide can induce the self-renewal and osteogenic differentiation of Nfatc1+MSCs through the activation of Ca2+ / Nfatc1 mediated by Reck / Gpr124 signalosomes, thereby exerting an osteogenic effect in vitro and in vivo ( Figure 6 E).

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

[0122] This example analyzes the mitigation of disease progression and the osteogenic effects of WNT7B-derived peptide 1797 in a miniature pig aging-related osteoporosis model. The specific experimental process is as follows:

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

[0124] (2) Minipigs received pelleted commercial food twice daily and had free access to water;

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

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

[0127] (5) Minipigs were intraperitoneally injected with 1797 peptide daily starting at age 6 for three consecutive months. After three months, the animals were euthanized and femoral tissues were obtained for CT analysis and reconstruction;

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

[0129] This example demonstrates that the application of WNT7B-derived polypeptide 1797 in miniature pigs with aging-related osteoporosis can effectively alleviate disease symptoms and increase bone mass.

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

[0131] In this example, WNT7B-derived peptide 1797 was used in a miniature pig extreme bone defect model to observe the bone regeneration effect and animal recovery effect. The specific experimental process is as follows:

[0132] (1) Establishment of critical femoral fracture model using miniature pigs:

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

[0134] b. According to ethical standards, miniature pigs should be euthanized within 6 months after stent implantation. Separate the femoral condyle and cut the skin and muscle with a scalpel and scissors. Use a circular saw to remove the intact condyle of the femur;

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

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

[0137] e. Wipe the surgical site with 4% chlorhexidine gluconate surgical scrub for surface disinfection, followed by a longitudinal incision in the skin on the medial side of the right knee;

[0138] f. Cut the subcutaneous tissue and periosteum to expose the surface of the femoral condyle, and use a bone trephine drill at a speed of 2000 revolutions per minute under saline irrigation (0.9% NaCl) to prepare a cavity. The exposed bone cavity of each animal was 10 mm in diameter and 6 mm in depth;

[0139] (2) The 1797 polypeptide scaffold was transplanted into the femoral cavity, and micro-CT analysis was performed 6 months after transplantation. Figure 8The data of μCT showed that the 1797 polypeptide scaffold group had a large amount of new bone, while the control group had no obvious regenerated bone tissue; the results of histological staining showed that 6 months after transplantation, the control group had obvious rejection reaction and was full of non-mineralized fibrous tissue, while the defect area of ​​the 1797 polypeptide scaffold group had been occupied by new bone, and the scaffold and the host bone had been integrated into a whole, and a large amount of new bone was also regenerated in the scaffold holes, indicating that in the microenvironment where the 1797 polypeptide existed, stem cells proliferated and differentiated into osteoblasts. Functional activity tests showed that the 1797 polypeptide scaffold treatment restored almost normal activities in miniature pigs with bone defects, while the control group still had poor activities. This example illustrates that the application of 1797 polypeptide in the miniature pig critical bone defect model can effectively promote new bone formation, restore the normal activity ability of animals, and has good biocompatibility.

[0140] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. An isolated polypeptide or a variant thereof, characterized in that The polypeptide consists of amino acids 200-217 and 323-336 of the WNT7B protein. The variant differs from the polypeptide from which it is derived in the substitution, deletion and / or addition of one or more amino acid residues, and retains the biological function of the polypeptide from which it is derived.

2. The isolated polypeptide or variant thereof according to claim 1, characterized in that The WNT7B protein has an amino acid sequence as shown in SEQ ID NO: 1, and the isolated polypeptide comprises an amino acid sequence as shown in SEQ ID NO:

4.

3. The isolated polypeptide or variant thereof according to claim 1, characterized in that The variants comprise the amino acid sequences shown in SEQ ID NOs: 5-7.

4. A fusion protein, characterized in that Comprising the isolated polypeptide or variant thereof according to any one of claims 1 to 3 and another polypeptide; Further, the additional polypeptide is optionally connected to the N-terminus or C-terminus of the polypeptide or its variant via a linker; Further, the additional polypeptide is selected from a protein tag, a targeting moiety or any combination thereof.

5. An isolated nucleic acid molecule, characterized in that The method comprises a nucleotide sequence encoding the isolated polypeptide or variant thereof according to any one of claims 1 to 3 or the fusion protein according to claim 4.

6. A carrier, characterized in that Comprising the isolated nucleic acid molecule according to claim 5.

7. A host cell, characterized in that Comprising the isolated nucleic acid molecule according to claim 5 or the vector according to claim 6.

8. A pharmaceutical composition, characterized in that Comprising the isolated polypeptide or variant thereof according to any one of claims 1 to 3, the fusion protein according to claim 4, the isolated nucleic acid molecule according to claim 5, the vector according to claim 6 or the host cell according to claim 7, and a pharmaceutically acceptable carrier and / or excipient.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition optionally further comprises an additional pharmaceutically active agent; Furthermore, the additional pharmaceutically active agent is other drugs that have the effect of promoting bone formation, bone regeneration and / or bone repair.

10. Use of the isolated polypeptide or variant thereof according to any one of claims 1 to 3, the fusion protein according to claim 4, the isolated nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the pharmaceutical composition according to claim 8 or 9 in the preparation of a drug, characterized in that: The medicament is used for promoting bone formation, bone regeneration, bone repair and / or preventing and / or treating osteoporosis.

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