A method for constructing a spontaneous ankylosing spondylitis model and its application

Abstract

The present invention discloses a method for constructing a spontaneous ankylosing spondylitis model and its application. The ankylosing spondylitis model is a non-human animal model, and the spontaneous ankylosing spondylitis non-human animal model is constructed by expressing mutant BMP9 protein. The present invention also provides a method for identifying a therapeutic agent for treating ankylosing spondylitis using the spontaneous ankylosing spondylitis non-human animal model. The present invention further provides an application of the spontaneous ankylosing spondylitis non-human animal model in evaluating the efficacy of a therapeutic agent in treating or preventing ankylosing spondylitis.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and more specifically, relates to a method for constructing a spontaneous ankylosing spondylitis model and its application. Background Art

[0002] Ankylosing spondylitis (AS) is a systemic disease characterized by chronic inflammation of the axial joints, primarily affecting the sacroiliac and spinal joints. In its late stages, it can lead to joint deformities and "bamboo-like" changes in the spine, resulting in severe physical deformity and disability. The prevalence of AS in my country is approximately 0.3%, with an estimated number of patients exceeding 4 million. AS typically develops in young adults (20-40 years old). The average age of onset in Chinese patients is 29.2 years, with a higher prevalence in men than in women (male:female = 2.8:1). AS is a chronic, progressive disease, and existing medications are incapable of halting its progression. Once patients develop the disease, they often gradually lose their ability to work within a decade. The early onset, high incidence, and high degree of disability of AS bring immense suffering to patients and their families, and have become a significant health burden on Chinese society.

[0003] AS primarily affects the axial skeleton and joints, making human tissue difficult to obtain. Therefore, experimental animal models of AS are crucial for gaining a deeper understanding of pathogenesis and developing new therapeutic strategies. Over the past two decades, researchers have developed a variety of AS-related animal models, including HLA-B27 transgenic rat / mouse models, inflammation-related models, and ankylosing enthesitis models. These experimental animal models have made significant contributions to our understanding of the complex pathomechanisms of AS, but they still have drawbacks and limitations. For example, the HLA-B27 / hβ2m double transgenic rat, the most widely used AS animal model, develops spondylitis and hindpaw arthritis similar to human AS. However, these models are associated with a long modeling period (requiring continuous breeding until rats are 7-9 months of age), a low success rate (only 30-50% of rats develop spondylitis by 9 months of age), mild symptoms, and differences from clinical ankylosing spondylitis. To date, no animal model fully recapitulates the clinical manifestations and pathological progression of human AS. Therefore, it is crucial to accelerate the development of experimental animal models that spontaneously develop AS and more closely resemble the clinical presentation of patients. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for constructing a spontaneous ankylosing spondylitis animal model and its application.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A first aspect of the present invention provides a method for constructing a spontaneous ankylosing spondylitis non-human animal model, wherein the non-human animal model expresses a mutant BMP9 protein.

[0007] Furthermore, the non-human animal model refers to a non-human animal that has or exhibits characteristics of a disease or condition.

[0008] Furthermore, the amino acid sequence of the mutant BMP9 protein is shown in SEQ ID NO: 1.

[0009] Furthermore, the method for expressing mutant BMP9 protein in ankylosing spondylitis non-human animal model comprises the following steps: introducing a nucleic acid molecule encoding the mutant BMP9 protein into a single-cell embryo or embryonic stem cell of a non-human animal.

[0010] Furthermore, the method for expressing mutant BMP9 protein in ankylosing spondylitis non-human animal model comprises the following steps: introducing an expression vector containing a nucleic acid molecule of the mutant BMP9 protein into a single-cell embryo or embryonic stem cell of a non-human animal.

[0011] Furthermore, the nucleic acid molecule comprises the following sequence: a nucleotide sequence formed by mutation of nucleotide G to T at position 948 in the protein coding region (Coding sequence, CDS) of the wild-type BMP9 gene (gene ID: 2658).

[0012] Furthermore, the expression vector includes but is not limited to linear polynucleotides, plasmids and viral vectors.

[0013] Furthermore, the viral vector includes but is not limited to a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0014] Furthermore, the expression vector introduction method includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection or liposome transfection.

[0015] Furthermore, the non-human animal is a rodent.

[0016] Furthermore, the rodents include the following options:

[0017] 1) Crittidae, such as Crittidae, ...

[0018] 2) Cricetidae such as black-striped hamster, field mouse, and crested mouse;

[0019] 3) Rodentia, such as black rat, brown rat, gerbil, New World rat, Old World rat, Norwegian rat, Polynesian rat, tree rat, cotton rat, wood rat, stick rat, rice rat, kangaroo rat, climbing rat;

[0020] 4) Malvinas, such as the long-tailed giant rat, the South African pocket rat, the African giant rat, and the Malvinas white-tailed rat;

[0021] 5) Myrmecophaga, such as Myrmecophaga and Myrmecophaga;

[0022] 6) Mole-shaped rats such as moles, bamboo rats, and zokors;

[0023] 7) Echinopsidae such as spiny rats;

[0024] 8) Rock rats, such as African rock rats.

[0025] Furthermore, the non-human animal model of ankylosing spondylitis exhibits one or more of the following symptoms: scoliosis, sternal protrusion, joint stiffness, tail ankylosing, forelimb deformity, hind limb sacroiliac joint deformity, spinal bone hyperplasia, spinal bone fusion, significantly decreased bone density, and significantly reduced trabeculae.

[0026] The second aspect of the present invention provides a cell line, which is derived from the non-human animal model of ankylosing spondylitis expressing mutant BMP9 protein prepared by the construction method described in the first aspect of the present invention.

[0027] The third aspect of the present invention provides an embryonic stem cell, which is derived from the non-human animal model of ankylosing spondylitis prepared by the construction method described in the first aspect of the present invention.

[0028] A fourth aspect of the present invention provides a method for identifying a therapeutic agent for treating ankylosing spondylitis, the method comprising the following steps:

[0029] 1) Administering a pharmaceutical agent to a non-human animal with ankylosing spondylitis prepared by the aforementioned construction method.

[0030] 2) performing one or more assays to determine whether the agent has a therapeutic effect on one or more abnormal symptoms associated with ankylosing spondylitis.

[0031] 3) When the agent has a therapeutic effect on one or more abnormal symptoms associated with ankylosing spondylitis, the agent is identified as a therapeutic agent.

[0032] Furthermore, the therapeutic agents include nonsteroidal anti-inflammatory agents, hormone preparations, targeted small molecule preparations, proteasome inhibitors, immunosuppressants, tumor necrosis inhibitors, cytokines, activators of costimulatory molecules, and inhibitors of inhibitory molecules.

[0033] A fifth aspect of the present invention provides use of a non-human animal model of ankylosing spondylitis in screening drugs for treating or preventing ankylosing spondylitis. The non-human animal model of ankylosing spondylitis is a model prepared using the construction method described above.

[0034] Furthermore, the drug includes one or more pharmaceutically acceptable excipients.

[0035] Furthermore, the excipients include adhesives, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants.

[0036] A sixth aspect of the present invention provides the use of a non-human animal model of ankylosing spondylitis in evaluating the therapeutic effect of a product for treating ankylosing spondylitis, wherein the non-human animal model of ankylosing spondylitis is a model prepared using the construction method described above.

[0037] In a seventh aspect, the present invention provides the use of a mutant BMP9 protein or a nucleotide sequence synthesized therefrom in constructing a spontaneous ankylosing spondylitis non-human animal model, wherein the amino acid sequence of the mutant BMP9 protein is shown in SEQ ID NO: 1.

[0038] Furthermore, the nucleotide sequence of the synthetic mutant BMP9 protein comprises the following sequence: a nucleotide sequence formed by mutation of the 948th nucleotide in the CDS sequence of the wild-type BMP9 gene from G to T.

[0039] Beneficial effects of the present invention:

[0040] The present invention provides a non-human animal model of ankylosing spondylitis. The model has a clear genetic background and is closer to the actual symptoms of the disease. It solves the problem that existing animal models are quite different from clinical diseases and is of great significance for the clinical research of ankylosing spondylitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the translation, cleavage, maturation process and mutation location of BMP9 protein; A: BMP9 protein translation, cleavage, and maturation process; B: Different mutation locations of BMP9;

[0042] Figure 2 The figure shows the expression of BMP9 protein by six mutant plasmids detected by Western blot. A: Expression of BMP9 protein by six mutant plasmids in cell lysate; B: Expression of BMP9 protein by six mutant plasmids in cell supernatant.

[0043] Figure 3The genotype of Tg-BMP9-MUT rats identified by PCR; NC: negative control; PC: positive control; M: nucleic acid marker; BLK: blank control; Tg-MUT: Tg-BMP9-MUT mutant transgenic positive rats;

[0044] Figure 4 This is the result of first-generation sequencing of the Tg-BMP9-MUT rat gene, where the red arrow points to the mutation site BMP9c.948G>T, p.Arg316Ser;

[0045] Figure 5 Figure 1 is the Western Blot results of BMP9 expression in lung tissue and liver tissue of WT rats and Tg-BMP9-MUT rats, where A: Western Blot results of lung tissue of two rats, B: Western Blot results of liver tissue of two rats;

[0046] Figure 6 These are back views of WT rats, BMP9 knockout rats (BMP9-KO), wild-type BMP9 overexpressing rats (Tg-BMP9), and Tg-BMP9-MUT overexpressing male rats, where A: WT rats, B: BMP9 knockout rats (BMP9-KO), C: wild-type BMP9 overexpressing rats (Tg-BMP9), and D: Tg-BMP9-MUT overexpressing male rats;

[0047] Figure 7 These are chest images of WT rats and Tg-BMP9-MUT overexpressing male rats, where A: WT rats, B: Tg-BMP9-MUT overexpressing male rats;

[0048] Figure 8 Figures are of the tails of WT rats and Tg-BMP9-MUT overexpressing male rats, where A: WT rats, B: Tg-BMP9-MUT overexpressing male rats;

[0049] Figure 9 Figures are of the feet of WT rats and Tg-BMP9-MUT overexpressing male rats, where A: WT rats, B: Tg-BMP9-MUT overexpressing male rats;

[0050] Figure 10 The following are side views of whole-body skeletal CT 3D models of WT rats and Tg-BMP9-MUT overexpressing male rats, where A: WT rats, B: Tg-BMP9-MUT overexpressing male rats;

[0051] Figure 11These are top views of whole-body skeletal CT 3D models of WT rats and Tg-BMP9-MUT-overexpressing male rats, where A: WT rats, B: Tg-BMP9-MUT-overexpressing male rats;

[0052] Figure 12 3D CT models of the spine of WT rats and Tg-BMP9-MUT overexpressing male rats, where A: WT rats, B: Tg-BMP9-MUT overexpressing male rats;

[0053] Figure 13 3D imaging of tibial trabeculae in WT rats and Tg-BMP9-MUT overexpressing male rats, where A: WT rats, B: Tg-BMP9-MUT overexpressing male rats. DETAILED DESCRIPTION

[0054] The present invention will be described in further detail below. It should be understood that the terms are intended to describe rather than limit the present invention.

[0055] The terms "nucleic acid sequence" or "polynucleotide" are used interchangeably herein and refer to a nucleic acid molecule, DNA or RNA, containing deoxyribonucleotides or ribonucleotides, respectively. A nucleic acid can be double-stranded, single-stranded, or contain portions of double-stranded or single-stranded sequences.

[0056] The term "expression" refers to the process by which a polynucleic acid is transcribed into mRNA and translated into a peptide, polypeptide or protein. If the polynucleic acid is derived from genomic DNA and a suitable eukaryotic host cell or organism is selected, expression may include splicing of the mRNA.

[0057] The term "nucleic acid" includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), which may be complementary DNA (cDNA) or genomic DNA.

[0058] The term "treat" refers to the administration of a compound or composition to control the progression of a disease. Control of disease progression is understood to mean achieving a beneficial or desired clinical outcome, including but not limited to alleviation of symptoms, reduction in disease duration, stabilization of the pathological state (particularly avoiding additional exacerbations), delay of disease progression, amelioration of the pathological state, and remission (partial or complete). Control of disease progression also involves prolongation of survival compared to expected survival without treatment.

[0059] The present invention provides a novel non-human animal model of ankylosing spondylitis. In fact, the inventors have discovered a new animal model of ankylosing spondylitis that reproduces the core features of the human disease.

[0060] The term "non-human animal" includes non-human vertebrates, more preferably mammals, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic class Rodentia (e.g., mice, rats, squirrels, beavers, woodchucks, gophers, voles, groundhogs, hamsters, guinea pigs, and agouti), including any descendants derived therefrom.

[0061] The term "non-human animal model" refers to a non-human animal that has or displays characteristics of a disease or condition. Use as an animal model refers to any use of an animal to study a disease or condition, such as to study progression or development or response to new or existing therapies.

[0062] The gene for bone morphogenetic protein 9 (BMP9, also known as growth differentiation factor 2, GDF2) is located on chromosome 10q11.22, with gene ID 2658. This includes the gene, its encoded protein, homologs, and mutations. The term "BMP9" encompasses the full-length, unprocessed gene or protein, as well as any form of the gene or protein derived from cellular processing. The term also encompasses naturally occurring variants of the biomarker. Gene IDs can be found at https: / / www.ncbi.nlm.nih.gov / gene / .

[0063] The mutant BMP9 protein refers to a protein expressing the amino acid sequence shown in SEQ ID NO: 2. The nucleic acid molecule for synthesizing the mutant BMP9 protein is formed by changing the 948th base of the wild-type BMP9 CDS sequence from G to T. The mutant BMP9 is a human mutant and is artificially synthesized.

[0064] As used herein, the term "expression vector" refers to a vector that may contain regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature, and such expression vectors may be used alone or in combination. Those skilled in the art are well aware of the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells containing the nucleic acid fragments of the mutant genes described herein. The expression vector may contain any combination of deoxyribonucleotides, ribonucleotides, or modified nucleotides, the expression vector may be transcribed to form RNA, wherein the RNA may be capable of forming double-stranded RNA and / or a hairpin structure, the expression vector may be expressed in a cell, or isolated, or synthesized, and the expression vector may further contain a promoter or other sequences that aid in the manipulation or expression of the construct.

[0065] In some embodiments, the nucleic acid sequence is operably linked to an expression vector. The expression vector is currently available through commercial channels, such as some viral vectors, plasmids, and bacteriophages.

[0066] In a preferred embodiment, the expression vector is a plasmid selected from conventional plasmids used in the art for constructing transgenic constructs. Typically, the plasmid contains a "spacer sequence" and multiple cloning sites or replacement sequences flanking the "spacer sequence," allowing the DNA sequence corresponding to the gene to be inserted into the multiple cloning site or to replace the replacement sequence in the multiple cloning site in both forward and reverse directions. The expression vector also typically contains a promoter, an origin of replication, and / or a marker gene.

[0067] In a specific embodiment of the present invention, the plasmid is pcDNA3.1-ALB.

[0068] As an alternative embodiment, the expression vector can be introduced into cells using known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, and binding to a cell membrane permeable peptide.

[0069] In a specific embodiment of the present invention, the expression vector is introduced into cells using microinjection.

[0070] As an alternative embodiment, the non-human animal is a rodent.

[0071] In some embodiments, rodents of the present disclosure include mice, rats, and hamsters as non-limiting examples. In some embodiments, rodents of the present disclosure include mice and rats as non-limiting examples. In some embodiments, rodents are selected from the superfamily Muroidea. In some embodiments, the rodent of the present disclosure is from a family selected from the group consisting of: Calomyscidae (e.g., Calomys hamster, Barth's hamster, Goodman's hamster, Golden hamster, Hall's hamster, Bearded hamster, Chu's hamster, Ulati hamster), Cricetidae (e.g., black-striped hamster, field mouse, crested hamster), Muridae (black rat, brown rat, gerbil, New World rat, Old World rat, Norway rat, Polynesian rat, tree rat, cotton rat, wood rat, stick rat, rice rat, kangaroo rat, climbing rat), Nesomyidae (long-tailed giant rat, South African gopher rat, African giant rat, Malvina white-tailed rat), Platacanthomyidae (e.g., spiny rat, pig-tailed rat), and Spalacidae (e.g., mole rat, bamboo rat, and zokor). In some embodiments, the rodent of the present disclosure is selected from a mouse or rat (family Muridae), a gerbil, a spiny rat, and a crested rat. In some embodiments, the rat of the present disclosure is from a member of the family Muridae.

[0072] In a specific embodiment of the invention, the rodent is a rat.

[0073] In some embodiments, the non-human animal model of ankylosing spondylitis exhibits one or more symptoms of scoliosis, sternal protrusion, joint stiffness, tail ankylosing, forelimb deformity, hind limb sacroiliac joint deformity, spinal bone hyperplasia, spinal bone fusion, significantly decreased bone density, and significantly decreased trabeculae.

[0074] In a specific embodiment of the present invention, the non-human animal with ankylosing spondylitis spontaneously develops ankylosing spondylitis after a period of growth.

[0075] The non-human animals of the present invention can be used for in vivo testing. Additionally, the non-human animals of the present invention can be used as a source of somatic, fetal, or embryonic cells that, once isolated and cultured, can be used for in vitro testing. Furthermore, if desired, immortalized cell lines can be prepared from these cells using conventional techniques. Thus, in another aspect, the present invention provides isolated cell lines derived from the non-human animals of the present invention.

[0076] In some embodiments, the present invention provides embryonic stem cells derived from the aforementioned non-human animal with ankylosing spondylitis.

[0077] In some embodiments, the present invention provides offspring of non-human animals. Offspring of non-human animals of the present invention can be obtained by conventional methods, for example, by conventional methods such as classical hybridization techniques between non-human animals of the present invention, or by in vitro fertilization of eggs and / or sperm of non-human animals of the present invention. As used herein, the term "offspring" refers to each offspring of each generation subsequent to the original transformed non-human animal.

[0078] In some embodiments, a non-human animal modified with a mutant BMP9 protein is bred with a wild-type animal to obtain mutant BMP9-positive animal offspring.

[0079] In some embodiments, the present invention provides a method of identifying a therapeutic agent for treating ankylosing spondylitis, the method comprising:

[0080] administering a pharmaceutical agent to the aforementioned non-human animal suffering from ankylosing spondylitis;

[0081] performing one or more assays to determine whether the agent has an effect on one or more abnormalities associated with ankylosing spondylitis;

[0082] An agent is identified as a therapeutic agent when the agent has a therapeutic effect on one or more disorders associated with ankylosing spondylitis.

[0083] The medicament of the present invention is preferably administered in a pharmaceutically acceptable vehicle. Suitable pharmaceutical carriers are known to those skilled in the art. For parenteral administration, the compound is typically dissolved or suspended in sterile water or saline. For enteral administration, the compound is incorporated into an inert carrier in the form of a tablet, liquid, or capsule. Suitable carriers can be starch or sugar, and include lubricants, flavorings, adhesives, and other materials of the same nature. The compound can also be administered topically via topical solutions, creams, gels, or polymeric materials (e.g., Pluronic™, BASF).

[0084] Alternatively, the compound can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to patients are well known to those skilled in the art. In essence, the material is dissolved in an aqueous solution, and if necessary, appropriate phospholipids and lipids are added together with a surfactant, and the material is dialyzed or ultrasonically treated as needed. Microspheres formed from polymers or proteins are well known to those skilled in the art and can be customized to directly enter the bloodstream through the gastrointestinal tract. Alternatively, the compound can be incorporated and the microspheres or microsphere complexes can be implanted to slowly release over a period of time ranging from several days to several months.

[0085] The methods of the present invention are preferably used to identify agents that alleviate such symptoms or signs.

[0086] In another embodiment, the invention relates to a method for evaluating the therapeutic effect of ankylosing spondylitis, comprising the steps of:

[0087] 1) providing a pharmaceutical composition or compound to be tested to a non-human animal model according to the present invention;

[0088] 2) Evaluate the effects observed on said model treated with the pharmaceutical composition or compound.

[0089] According to a preferred embodiment of the present invention, the effect to be observed refers to physiological and pathological changes. The physiological and pathological changes to be detected in the animal model of the present invention refer to any improvement of the physiological and pathological changes existing in the animal model as previously described.

[0090] In another embodiment, the present invention provides use of the animal model according to the present invention, the cell line according to the present invention, or the embryonic stem cell according to the present invention in screening drugs for treating or preventing ankylosing spondylitis.

[0091] Candidate compounds or drugs for use in the methods of the present invention may include all different types of organic or inorganic molecules, including peptides, oligosaccharides or polysaccharides, fatty acids, steroids, etc. In addition, possible compounds to be screened include, for example, hematopoietic stem cells, enzymes, and gene therapy products, such as recombinant vectors, etc. These compounds can be administered alone or in combination with each other.

[0092] Screening using animal models can involve administering candidate compounds before, during, or after the development of a specific disease phenotype. Symptoms of disease progression or regression can be monitored using diagnostic tests known to those skilled in the art. Methods for monitoring symptoms of disease progression or regression are well known to those skilled in the art, such as Doppler ultrasound, pathological examination, and the like.

[0093] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0094] Example 1 Vector transfection and detection of mutant proteins

[0095] 1. Vector Construction

[0096] Seven BMP9 eukaryotic expression vectors were constructed, including a BMP9 wild-type plasmid and six mutant plasmids (p.R316S, S320C, V109L, S282framshift (frameshift mutation), A353T and V423M), which were transfected into eukaryotic cells HEK293EBNA. The cells themselves and cell supernatant were harvested, and the expression of mutant proteins was detected using BMP9 antibodies.

[0097] 2. Results

[0098] The BMP9 protein is 429 amino acids in length and contains three domains: the signal peptide domain at the N-terminus (Signalpeptide, amino acids 1-22), the pro-domain in the middle (Pro-domain, amino acids 23-319) and the mature BMP9 at the C-terminus (Mature-BMP9, amino acids 320-429). After the Pre-Pro-BMP9 full-length protein is translated in vivo, it is secreted out of the cell under the guidance of the signal peptide. The originally connected Pro-domain and Mature-BMP9 are cut apart by the Fusion enzyme at the 316-319 amino acid position (RXXR), and then covalently bonded to form a dimer, which is transported throughout the body through the blood circulation. Figure 1 As shown in A.

[0099] The amino acids 316-319 (RXXR) of the BMP9 protein are highly conserved among all BMP family proteins and are very important for protein cleavage, maturation, and normal function. The BMP9 mutation p.R316S that this patent focuses on is located at the first position of the "RXXR" domain, which converts "R (arginine)" to "S (serine)". Figure 1 As shown in B.

[0100] To clarify the effect of the BMP9 mutation p.R316S on protein expression, we constructed eukaryotic expression vectors containing R316S and five other BMP9 mutations (S320C, V109L, S282frameshift (frameshift mutation), A353T, and V423M) and transformed them into 293 cells. Western blot analysis of BMP9 protein expression in cell supernatants revealed that no mature-BMP9 protein was detected in the supernatants of the six mutant plasmids. Figure 2 As shown in the blue box of A, R316S expresses a large amount of uncut mutant proteins. Figure 2 The red framed part of B indicates that the R316S mutation not only reduces the Mature-BMP9, but also produces a large amount of exogenous, uncut mutant protein.

[0101] Example 2 Construction and detection of spontaneous ankylosing spondylitis model

[0102] 1. Construction of vector

[0103] 1. Artificially synthesize the full-length CDS of human mutant BMP9; this mutant has only one nucleotide difference, c.G948T, in the CDS sequence compared with wild-type BMP9 (GeneID: 2658).

[0104] 2. A transgenic plasmid containing the full-length CDS of a human mutant BMP was constructed and named pcDNA3.1-ALB-BMP9-MUT. This plasmid was independently constructed in the laboratory and uses the ALB gene promoter sequence to drive high expression of the human mutant BMP9 gene in the liver.

[0105] 3. The full length of pcDNA3.1-ALB-BMP9-MUT plasmid is 8574 bp, and the nucleic acid was confirmed to be correct by Sanger sequencing.

[0106] 4. The amino acid sequence expressed by the human mutant BMP9 is shown in SEQ ID NO: 1, and the specific mutation is that the amino acid at position 316 is changed from arginine to serine.

[0107] 2. Microinjection

[0108] 1. Vasectomy male rats: SD male rats with vasectomy.

[0109] 2. Superovulation: 10 3-4 week old SD mice were injected with hormones for superovulation.

[0110] 3. Fertilized egg injection: Take about 100 fertilized eggs for injection.

[0111] 4. Preparation of recipient mice: 8-week-old SD female mice were mated with ligated male mice and the female mice with visible thrombosis were selected.

[0112] 5. Embryo transplantation: The injected fertilized egg is transplanted into the ampulla of the fallopian tube of the recipient mouse.

[0113] 3. Genotype identification

[0114] 1. Tail cutting and numbering: For rats born 7-10 days old, cut off the toes and tail tips for numbering.

[0115] 2. Genomic DNA extraction: Use the genomic DNA extraction kit (EE101-12) from Transgen to extract rat genomic DNA.

[0116] 3. PCR detection: Synthesize PCR genotyping primers (Table 1) and use the TaKaRa RR042A kit according to the reagent ratios shown in Table 2 and the reaction conditions shown in Table 3 to perform genotyping on rats.

[0117] Table 1 Primer sequences for identifying Tg-BMP9-MUT genotype

[0118] R-BMP9-Mut-F 5'-TCCAGATGGCAAACATACGC-3' SEQ ID NO:2 R-BMP9-Mut-R 5'-GCTCCACCCTTGTCTTATCCTG-3' SEQ ID NO:3 Destination fragment 554bp

[0119] Table 2 PCR reaction system for identifying Tg-BMP9-MUT genotype

[0120] <![CDATA[10×LA PCR BufferⅡ(Mg 2+ Plus)]]> 2.0 μl dNTP Mixture (2.5 μM) 1.6 μl Primer-S (50 μM) 0.2 μl Primer-A (50 μM) 0.2 μl Template DNA 1.0 μl LA Taq 0.2 μl <![CDATA[Add ddH2O to the total volume]]> 20.0μl

[0121] Table 3 PCR amplification procedure for identifying Tg-BMP9-MUT genotype

[0122]

[0123] 4. Result analysis:

[0124] After PCR was completed, the PCR reaction was terminated with 6× loading buffer and electrophoresis was performed on 1% agarose gel. Only rats carrying the BMP9 transgenic fragment could amplify a 554 bp positive band, while negative rats in the same litter had no band ( Figure 3 For the amplified PCR products, Sanger sequencing was used to verify the existence of the mutation site ( Figure 4 ), the mutation site is BMP9c.948G>T, p.Arg316Ser. Western Blot was used to detect the expression of BMP9 in lung and liver tissues of WT and Tg-BMP9-MUT rats ( Figure 5), endogenous BMP9 protein was detected in the lung and liver tissues of both rats (indicated by blue arrows), but a mutant BMP9 protein was expressed in the lung and liver tissues of Tg-BMP9-MUT rats (indicated by red arrows). This mutant BMP9 protein only existed in mutant rats and had a molecular weight of 60KD, which was significantly larger than the endogenous BMP9 protein (55KD).

[0125] 4. Breeding offspring:

[0126] 1. The obtained founder rats were used as F0 generation rats and mated with wild-type SD rats to obtain F1 generation, and F1 generation Tg-BMP9-MUT rats were obtained by PCR identification.

[0127] 2. In each generation, heterozygous positive male mice are mated with wild-type female mice in a ratio of 1:2 to obtain the next generation of rats.

[0128] 3. As of June 2022, the rats have been passed down to the F5 generation, and the genotype is stable. The proportion of positive rats in each generation is 23-83%, and the average positive rate is 50%, which is in line with Mendel's law of inheritance (Table 4).

[0129] Table 4 Statistical results of Tg-BMP9-MUT rat breeding

[0130]

[0131] 5. Phenotypic Analysis of Spontaneous Ankylosing Spondylitis in Tg-BMP9-MUT Rats

[0132] 1. Wild-type rats (WT), BMP9 knockout rats (BMP9-KO), wild-type BMP9 transgenic rats (Tg-BMP9), and Tg-BMP9-MUT male rats without any genetic modification have basically normal phenotypes before 1 month of age, with no obvious abnormalities in the spine or bones.

[0133] 2. Spontaneous bone abnormalities gradually appeared in Tg-BMP9-MUT male rats from 1.5 months of age. By 3 months of age, 100% of male Tg-BMP9-MUT rats had bone abnormalities ( Figure 6 D), WT wild-type rats without any genetic modification ( Figure 6 A), BMP9 knockout rats BMP9-KO ( Figure 6 B), wild-type BMP9 transgenic rats Tg-BMP9 ( Figure 6 C) showed no skeletal abnormalities. Therefore, in subsequent experiments, wild-type WT rats without any genetic modification were used as control mice.

[0134] Three-month-old WT wild-type rats without any genetic modification were used as the control group, and three-month-old male Tg-BMP9-MUT male rats were used as the experimental group. The following phenotypes were observed:

[0135] 1) The control group rats had no abnormalities in the spine and hind feet ( Figure 6 A); The rats in the experimental group had scoliosis and right hind foot valgus ( Figure 6 D).

[0136] 2) There was no abnormality in the sternum of the rats in the control group ( Figure 7 A); The sternum of the rats in the experimental group was prominent, and the left front leg and right hind leg could not be bent ( Figure 7 B).

[0137] 3) The tails of the rats in the control group showed no abnormalities ( Figure 8 A); The tails of rats in the experimental group were stiff and could not droop naturally ( Figure 8 B).

[0138] 4) The hind legs of the rats in the control group showed no abnormalities and the joints were flexible ( Figure 9 A); The hind legs of rats in the experimental group were swollen and the joints were stiff ( Figure 9 B).

[0139] 3. 3-month-old WT wild-type rats without any genetic modification were used as the control group, and 3-month-old male Tg-BMP9-MUT male rats were used as the experimental group. The CT examination results were consistent with the abnormal phenotype observed in appearance.

[0140] Whole-body skeletal CT 3D modeling showed that the skeleton of the rats in the control group was normal, and the limbs were bent normally after anesthesia ( Figure 10 A); The rats in the experimental group had bulging spines, deformed forelimbs, and deformed sacroiliac joints of the hindlimbs. After anesthesia, the right forelimbs and hindlimbs could not bend normally ( Figure 10 B).

[0141] CT 3D modeling from a top-down angle showed that the spine of the rats in the control group was normal ( Figure 11 A); The spinal column of the rats in the experimental group was obviously scoliotic ( Figure 11 B).

[0142] The spinal column local fine CT examination showed that the spinal column of the control group rats was normal ( Figure 12 A); The rats in the experimental group had spinal bone hyperplasia, bone fusion, and typical "bamboo-like" lesions ( Figure 12 B).

[0143] 4. 3-month-old WT wild-type rats without any genetic modification were used as the control group, and 3-month-old male Tg-BMP9-MUT male rats were used as the experimental group. 3D imaging analysis of the rat tibial trabeculae was performed. The analysis results showed that the bone density of the Tg-BMP9-MUT rats in the experimental group was significantly reduced compared with the control group ( Figure 13 ).

[0144] The results of quantitative bone density analysis are shown in Table 5. The tibia bone density of the experimental group Tg-BMP9-MUT rats was only 52% of that of the tibia in the control group WT rats (158±40 vs 304±8, p<0.001), the femur bone density of the experimental group Tg-BMP9-MUT rats was only 28% of that of the femur in the control group WT rats (63±25 vs 229±12, p<0.001), and the trabecular number of the experimental group Tg-BMP9-MUT rats was only 69% of that of the trabecular number of the control group WT rats (P<0.05), indicating that the bone density and trabeculae of the experimental group Tg-BMP9-MUT rats were significantly reduced.

[0145] Table 5 Tibial bone density, femoral bone density, and trabecular number test results

[0146]

[0147]

[0148] Note: sh: tibia; th: femur; sp: spine; BMD: bone mineral density; BVF: bone volume fraction; BS / BV: bone surface area to bone volume ratio; Tb.Th: trabecular thickness; Tb.Nub: trabecular number; Tb.Sp: trabecular spacing; Tb.PF: trabecular pattern factor

[0149] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a non-human animal model of spontaneous ankylosing spondylitis, characterized in that, The non-human animal model of ankylosing spondylitis expresses mutant BMP9 protein; The mutant BMP9 protein is such that the amino acid residue corresponding to the 316th position in the wild-type BMP9 amino acid sequence is mutated from arginine to serine.

2. The construction method according to claim 1, wherein The amino acid sequence of the mutant BMP9 protein is as shown in SEQ ID NO:

1.

3. The construction method according to claim 1, wherein The non-human animal model refers to a non-human animal having or showing the characteristics of a disease or medical condition.

4. The construction method according to claim 1, wherein The method for the non-human animal model of ankylosing spondylitis to express mutant BMP9 protein comprises the following steps: introducing a nucleic acid molecule encoding the mutant BMP9 protein into a single-cell embryo or embryonic stem cell of a non-human animal.

5. The construction method according to claim 4, characterized in that, The nucleic acid molecule comprises the following sequence: the nucleotide sequence formed by mutating the 948th nucleotide on the CDS sequence of the wild-type BMP9 gene from G to T.

6. The construction method according to claim 1, characterized in that The method for the non-human animal model of ankylosing spondylitis to express mutant BMP9 protein comprises the following steps: introducing an expression vector containing the nucleic acid molecule of the mutant BMP9 protein into a single-cell embryo or embryonic stem cell of a non-human animal.

7. The construction method according to claim 6, characterized in that The nucleic acid molecule comprises the following sequence: the nucleotide sequence formed by mutating the 948th nucleotide on the CDS sequence of the wild-type BMP9 gene from G to T.

8. The construction method according to claim 6, wherein The expression vector includes linear polynucleotides, plasmids and viral vectors.

9. The construction method according to claim 8, wherein The viral vectors include lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors.

10. The construction method according to claim 6, characterized in that, The method for introducing the expression vector includes electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection or liposome transfection.

11. The construction method according to claim 3, characterized in that The non-human animal is a rodent.

12. The construction method according to claim 11, characterized in that, The rodents include the following options: 1) Calomyscidae such as Calomyscus bailwardi, Calomyscus ballerus, Calomyscus canus, Calomyscus campestris, Calomyscus hotsoni, Calomyscus mystax, Calomyscus centralis, Calomyscus uralensis; 2) Cricetidae such as Cricetulus barabensis, Microtus fortis, Lophiomys imhausi; 3) Muridae such as Rattus rattus, Rattus norvegicus, Meriones unguiculatus, Rattus novegicus, Rattus rattus, Rattus norvegicus, Rattus exulans, Rattus rattus, Sigmodon hispidus, Neotoma micropus, Neotoma floridana, Neotoma mexicana, Oryzomys palustris, Dipodomys ordii, Vandeleuria oleracea; 4) Nesomyidae such as Hypogeomys antimena, Cricetomys gambianus, Thryonomys swinderianus, Macrotarsomys bastardi; 5) Platacanthomyidae such as Platacanthomys lasiurus, Typhlomys cinereus; 6) Spalacidae such as Talpa europaea, Rhizomys sinensis, Myospalax fontanieri; 7) Echimyidae such as Echimys chrysurus; 8) Petromuridae such as Petromus typicus.

13. The construction method according to claim 1, characterized in that The non-human animal model of ankylosing spondylitis exhibits one or more of the following symptoms: scoliosis, pectus carinatum, joint stiffness, tail rigidity, forelimb deformity, hindlimb sacroiliac joint deformity, spinal osteoproliferation, spinal bone fusion, significant reduction in bone density, significant reduction in trabecular bone.

14. A method for identifying a therapeutic agent for treating ankylosing spondylitis, characterized in that, The method comprises the following steps: 1) Administering a medicament to the non-human animal with ankylosing spondylitis prepared by the construction method according to any one of claims 1-13; 2) Conducting one or more assays to determine whether the medicament has a therapeutic effect on one or more abnormal symptoms related to ankylosing spondylitis; 3) When the medicament has a therapeutic effect on one or more abnormal symptoms related to ankylosing spondylitis, identifying the medicament as a therapeutic agent.

15. The method according to claim 14, wherein The therapeutic agent includes non-steroidal anti-inflammatory agents, hormonal agents, targeted small molecule agents, proteasome inhibitors, immunosuppressive agents, tumor necrosis inhibitors, cytokines, activators of co-stimulatory molecules, and inhibitors of inhibitory molecules.

16. Use of a non-human animal model of ankylosing spondylitis in screening for a drug for treating or preventing ankylosing spondylitis, characterized in that, The non-human animal model of ankylosing spondylitis is a model prepared by using the construction method described in any one of claims 1-13.

17. The method according to claim 16, wherein The drug includes one or more pharmaceutically acceptable excipients.

18. The method according to claim 17, wherein The excipients include binders, fillers, disintegrants, lubricants, ointments, preservatives, antioxidants, flavoring agents, fragrances, solubilizing agents, emulsifiers, solubilizers, osmotic pressure regulators, and coloring agents.

19. Use of a non-human animal model of ankylosing spondylitis in evaluating the therapeutic effect of a product for treating ankylosing spondylitis, characterized in that, The non-human animal model of ankylosing spondylitis is a model prepared by using the construction method described in any one of claims 1-13.

20. Use of the mutant BMP9 protein or the nucleotide sequence encoding the same as described in claim 1 in constructing a non-human animal model of ankylosing spondylitis, characterized in that, The mutant BMP9 protein has the amino acid residue corresponding to position 316 in the wild-type BMP9 amino acid sequence mutated from arginine to serine.

21. The application according to claim 20, wherein The amino acid sequence of the mutant BMP9 protein is as shown in SEQ ID NO:

1.

22. The application according to claim 20, characterized in that, The nucleotide sequence of the synthetic mutant BMP9 protein contains the following sequence: the nucleotide sequence formed by mutating the 948th nucleotide on the CDS sequence of the wild-type BMP9 gene from G to T.

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