A focal adhesion kinase mutant and its use in the preparation of a drug for preventing and / or treating osteoporosis

Abstract

This invention discloses a speckle adhesion protein mutant and its application in the preparation of drugs for the prevention and / or treatment of osteoporosis, relating to the field of biomedical technology. The invention provides a speckle adhesion protein mutant comprising a protein obtained by mutating the amino acid at position 219 of speckle adhesion protein from lysine to arginine. Based on the three shortcomings of existing technologies—single therapeutic target, lack of treatment strategies targeting specific mechanisms of osteoporosis in ankylosing spondylitis (AS), and failure to intervene in key pathological changes in the bone marrow microenvironment—this invention is the first to discover and demonstrate that lactation modification of lysine (K219) at position 219 of speckle adhesion protein (VCL) is a key molecular event mediating impaired osteogenic differentiation function of mesenchymal stem cells (MSCs) in inflammatory osteoporosis of ankylosing spondylitis (AS), providing a novel solution with great clinical translational potential for the treatment of inflammatory osteoporosis in AS.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a focal adhesion protein mutant and its application in the preparation of drugs for the prevention and / or treatment of osteoporosis. Background Technology

[0002] Ankylosing spondylitis (AS) is a chronic inflammatory autoimmune disease that primarily affects the axial skeleton, leading to sacroiliitis, spondylitis, and ligament osteophyte formation. In advanced stages, it can cause spinal ankylosis and joint fusion. Notably, approximately 25% of AS patients develop inflammatory osteoporosis during the disease's progression, characterized by decreased bone mineral density, bone microarchitectural deterioration, and a significantly increased risk of fractures, severely impacting their quality of life.

[0003] Currently, clinical treatment strategies for AS primarily focus on suppressing the systemic inflammatory response. First-line drugs are nonsteroidal anti-inflammatory drugs (NSAIDs) used to relieve pain and stiffness. For patients with high disease activity, biologics are used, such as tumor necrosis factor-α (TNF-α) inhibitors (e.g., etanercept, adalimumab) and interleukin-17A (IL-17A) inhibitors (e.g., secukinumab). These therapies effectively control inflammation, relieve symptoms, and slow the progression of spinal fusion. However, existing treatments for inflammatory osteoporosis associated with AS have significant limitations. While bisphosphonates (e.g., zoledronic acid) or nuclear factor κB receptor activator ligand (RANKL) inhibitors (e.g., denosumab) can be used to treat common osteoporosis, their efficacy in the inflammatory microenvironment of AS is unclear, and they were not developed specifically for the etiology of AS osteoporosis. In other words, there is currently a lack of effective drugs that can directly target the core pathogenesis of osteoporosis in AS. Existing drugs for treating osteoporosis in AS have the following shortcomings: (1) Single therapeutic target and insufficient improvement in bone metabolism: Existing biological agents mainly target inflammatory factors such as TNF-α or IL-17A. Although they can inhibit inflammation, their therapeutic effect on osteoblast dysfunction and bone formation inhibition caused directly by the chronic inflammatory microenvironment is limited. Inflammation and bone loss often coexist in AS, but their mechanisms do not completely overlap. Inhibiting inflammation cannot completely reverse the bone metabolic imbalance that has already occurred. (2) Lack of treatment plans targeting the specific mechanisms of osteoporosis in AS: The pathogenesis of inflammatory osteoporosis in AS is complex, involving multiple levels such as changes in the bone marrow microenvironment and dysfunction of mesenchymal stem cells (MSCs). Existing drugs are not designed based on this specific mechanism, so they cannot fundamentally correct the key pathological link of impaired osteogenic differentiation capacity of MSCs in AS, resulting in unsatisfactory treatment effects on osteoporosis and individual differences. (3) Failure to intervene in key pathological changes in the bone marrow microenvironment: chronic inflammation of AS leads to a large number of immune cells infiltrating the bone marrow, and their vigorous glycolytic metabolism causes a large accumulation of local lactic acid (up to 10-30 mM).

[0004] At the molecular level, vincristin (VCL), a key cytoskeleton protein, plays a crucial role in cell adhesion and mechanotransduction. VCL forms a "bridge" structure by connecting integrins to the actin cytoskeleton, stabilizing vincristin and activating downstream signaling pathways such as RhoA / ROCK, inducing the expression of osteogenic genes (e.g., RUNX2, Osterix), and promoting osteogenic differentiation of mesenchymal stem cells (MSCs). This mechanism is considered essential for maintaining bone metabolic homeostasis. Lactic acid, as a signaling molecule, catalyzes lactation modification of proteins, thereby regulating cellular function; however, the role of this modification in ankylosing spondylitis (AS) osteoporosis has not been considered or targeted in current treatment strategies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a focal adhesion protein mutant and its application in the preparation of drugs for the prevention and / or treatment of osteoporosis. It not only overcomes the three major shortcomings of the prior art, but also shows significant advantages in terms of the precision, specificity, fundamental nature and durability of the treatment strategy, providing a brand-new solution with great clinical translational potential for the treatment of inflammatory osteoporosis in AS.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a plaque protein mutant, wherein the mutant comprises a protein obtained by mutating the amino acid at position 219 of the plaque protein from lysine to arginine.

[0007] This invention addresses three shortcomings of existing technologies: limited therapeutic targets (insufficient improvement in bone metabolism), lack of treatment options targeting the specific mechanisms of osteoporosis in ankylosing spondylitis (AS), and failure to intervene in key pathological changes in the bone marrow microenvironment. For the first time, it discovers and confirms that lactation modification of lysine residue 219 (K219) of vitreous adhesion protein (VCL) is a key molecular event mediating impaired osteogenic differentiation of mesenchymal stem cells (MSCs) in inflammatory osteoporosis of ankylosing spondylitis (AS). By specifically targeting this key pathological step of lactation modification at the K219 site of VCL protein in the bone marrow microenvironment, this invention develops a treatment method that can directly block abnormal lactation modification, restore normal VCL function, and thus effectively promote MSC osteogenic differentiation and improve bone metabolism, overcoming the limitations of existing anti-inflammatory therapies in improving bone formation.

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

[0009] The present invention also provides a nucleotide sequence encoding the aforementioned focal adhesion protein mutant.

[0010] The present invention also provides an adeno-associated virus, including the aforementioned focal adhesion protein mutant.

[0011] The present invention also provides a method for constructing the adeno-associated virus, wherein the focal adhesion protein mutant is cloned into a plasmid vector, the focal adhesion protein mutant is expressed under the control of an MSC-specific promoter, and transfected into 293T cells, the virus is purified, and adeno-associated virus VCL-K219R AAV is obtained.

[0012] This invention designs and constructs a VCL-K219R point mutant that can specifically resist lactation modification, and successfully clones it into a recombinant adeno-associated virus (AAV) vector, using an MSC-specific promoter to achieve targeted expression.

[0013] Preferably, the vector is AAV9 serotype virus.

[0014] The inventors of this application have discovered that by using AAV9 serotype virus as a vector and taking advantage of its natural bone marrow tissue tropism, the therapeutic gene VCL-K219R can be efficiently delivered to bone marrow mesenchymal stem cells (MSCs) at the lesion site.

[0015] The present invention also provides a recombinant gene therapy vector comprising the aforementioned nucleotide sequence.

[0016] The present invention also provides the use of the aforementioned focal adhesion protein mutant, the aforementioned adeno-associated virus, or the aforementioned recombinant gene therapy vector in the preparation of medicaments for the prevention and / or treatment of osteoporosis.

[0017] The inventors of this application have discovered that overexpression of VCL-K219R can specifically reverse the lactation modification of VCL induced by a high lactate environment, restore cytoskeleton assembly and mechanical signal transduction, and ultimately significantly improve the osteogenic differentiation capacity of MSCs and increase bone formation.

[0018] In a preferred embodiment of the application described in this invention, the osteoporosis is inflammatory osteoporosis caused by ankylosing spondylitis.

[0019] As a preferred embodiment of the application described in this invention, the drug promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0020] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of ankylosing spondylitis-related inflammatory osteoporosis, the pharmaceutical composition comprising the aforementioned focal adhesion protein mutant, the aforementioned adeno-associated virus, or the aforementioned recombinant gene therapy vector.

[0021] Preferably, the adeno-associated virus titer is 1×10⁻⁶. 12 -5×10 12 vg / mL.

[0022] Preferably, the effective dose of the pharmaceutical composition is 1×10⁻⁶. 11 -5×10 11 vg / kg body weight.

[0023] More preferably, the pharmaceutical composition is administered via intravenous injection or local bone tissue injection.

[0024] As a preferred embodiment of the pharmaceutical composition of the present invention, it further includes excipients or pharmaceutically acceptable carriers.

[0025] The beneficial effects of the present invention: The present invention provides a focal adhesion protein mutant and its application in the preparation of drugs for the prevention and / or treatment of osteoporosis, which has the following advantages: (1) Precise targeting, directly targeting the core pathological link: The present invention for the first time precisely locates the therapeutic target to the K219 lactation modification of VCL protein. By designing the VCL-K219R mutant, it can specifically compete for and reverse the pathological lactation modification, directly correct the osteogenic dysfunction of MSC from the functional protein level, realize the fundamental transformation from "anti-inflammatory" to "bone formation", and solve the fundamental problem of insufficient improvement of bone metabolism in existing treatments; (2) Novel mechanism, strong specificity for AS osteoporosis: Existing anti-osteoporosis drugs (such as bisphosphonates) are not designed for the inflammatory microenvironment of AS, and generally have problems such as poor specificity and large side effects. This invention is designed based on the bone marrow-specific high lactate microenvironment of AS. The "lactate-VCL lactation-osteogenic inhibition" axis that is intervened in is a potential specific mechanism of AS inflammatory osteoporosis. It has stronger targeting and better expected efficacy in the treatment of AS complicated with osteoporosis, and avoids the side effects of the "pan-targeting" of traditional drugs; (3) It fundamentally intervenes in the pathological changes of the bone marrow microenvironment: Existing technologies cannot effectively intervene in the key pathological change of lactate accumulation in the local bone marrow microenvironment. The gene therapy strategy (AAV delivery of VCL-K219R) adopted in this invention is a method of "empowering" cells. Even in a high lactate environment, the VCL protein of genetically modified MSCs can resist abnormal modification and perform its normal function, thus overcoming the adverse effects of the microenvironment at the cellular level and achieving precise correction of the core link of the pathological microenvironment—cellular dysfunction. This is something that small molecule drugs and biological agents cannot achieve. (4) Long-lasting efficacy: Compared with existing drugs that require frequent administration (such as daily oral administration or weekly / monthly injection), once AAV-mediated gene therapy is successful, it can make target cells express the protective VCL-K219R protein for a long time (or even permanently), thereby achieving the goal of single treatment and long-term effectiveness. It is hoped that this will greatly improve patient compliance and reduce the economic burden of long-term treatment; (5) Double safety guarantee, high safety: The VCL-K219R mutant is only a single amino acid point mutation (K→R). This mutation simulates the naturally existing, non-lactable state and does not introduce exogenous active elements. It preserves the natural structure and function of VCL protein to the greatest extent and theoretically avoids the risk of introducing unknown functions; The use of MSC-specific promoters (such as osteocalcin promoters) can drive the expression of the target gene mainly in osteoblast lineage cells, realize tissue-specific targeting, limit the unnecessary expression of genes, and further enhance the safety of treatment. Attached Figure Description

[0026] Figure 1The effect of lactation modification on MSC osteogenicity, where A is the Western Blot (WB) verification of the level of lactation modification; B is the result of Alizarin Red S (ARS) staining.

[0027] Figure 2 The results of bioinformatics analysis of lactation modification at the VCL-K219 site.

[0028] Figure 3 To reverse bone loss in SKG mice by overexpressing VCL-K219R via AAV, Figure A shows the results of Western blotting of proteins extracted from femoral bone marrow cells; Figure B shows the quantitative analysis results. Detailed Implementation

[0029] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0030] Example 1 This embodiment experimentally verifies the effect of lactation modification on MSC osteogenicity. The specific experimental steps are as follows: 1. Obtaining and culturing human bone marrow-derived MSCs: Human bone marrow mesenchymal stem cells (MSCs) are obtained and cultured through the following methods: Bone marrow sample source: After approval by the ethics committee and obtaining informed consent, 3-5 mL of bone marrow fluid was collected from the posterior superior iliac crest of healthy volunteers or AS patients (using heparin anticoagulation).

[0031] Separation method: Density gradient centrifugation was used. Bone marrow fluid was diluted with an equal volume of PBS and slowly added to human lymphocyte separation medium (Ficoll-Paque PLUS, density 1.077 g / mL). The mixture was centrifuged at 800×g for 30 minutes (room temperature, ascending speed 9, descending speed 6). Mononuclear cells from the white membrane layer were aspirated and washed twice with PBS (centrifuged at 300×g for 10 minutes).

[0032] Culture and expansion: Cells were resuspended in complete culture medium (α-MEM medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM L-glutamine) and cultured at 5 × 10⁻⁶ cells / mL. 5 Inoculate the culture flasks at a density of cells / cm² and incubate them in a 37°C, 5% CO2 incubator.

[0033] Passaging and identification: Change the medium every 3-4 days. When the cells reach 80-90% confluence, digest them with 0.25% trypsin-EDTA for passage. Take the third-generation cells and detect surface markers by flow cytometry (CD73, CD90, CD105 positive rate >95%; CD34, CD45, HLA-DR negative rate <5%) and perform osteogenic and adipogenic differentiation induction to identify them as MSCs.

[0034] 2. Sodium lactate treatment to simulate an inflammatory microenvironment: To simulate the high lactate state in the bone marrow microenvironment of AS inflammation, MSCs were treated with sodium lactate: Sodium lactate solution preparation: 1 M sodium lactate stock solution was prepared with PBS, filtered sterilized, and aliquoted at -20℃. Treatment concentration and time: MSCs in good growth condition from passages 3-5 were treated with osteogenic induction medium containing 10 mM sodium lactate (complete medium supplemented with 50 μM ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone). A control group (normal osteogenic induction medium) was also established. Treatment continued throughout the entire osteogenic induction cycle (21 days) for functional assays.

[0035] 3. Western Blot (WB) to verify the level of lactation modification: To determine the lactation modification level of proteins in MSCs after lactate treatment, Western blotting was used. The specific steps are as follows: (1) Protein sample preparation: After treatment, cells were washed twice with pre-cooled PBS, and lysed on ice for 30 minutes with RIPA lysis buffer (containing 1 mM PMSF and 1× protease inhibitor cocktail). The cells were centrifuged at 12000×g for 15 minutes at 4℃, and the supernatant was collected. The protein concentration was determined by the BCA method.

[0036] (2) Electrophoresis and transfer: 30 μg of total protein was subjected to 10% SDS-PAGE gel electrophoresis (80V constant voltage for 30 minutes, followed by 120V for 60-90 minutes). The protein was then transferred to a PVDF membrane by wet transfer (300 mA constant current for 90 minutes).

[0037] (3) Blocking and primary antibody incubation: Block with TBST blocking buffer containing 5% skim milk at room temperature for 1 hour. Incubate with primary antibody overnight at 4°C. Anti-pan-lactic acidification modified antibody (Anti-Pan-Kla antibody, such as PTM-1401RM, dilution 1:1000) and anti-β-actin antibody (such as AC004, dilution 1:5000) were used as internal controls.

[0038] (4) Secondary antibody incubation and development: Incubate with HRP-labeled secondary antibody of the corresponding species (1:5000 dilution) at room temperature for 1 hour. Develop with ECL chemiluminescence reagent and acquire images on a chemiluminescence imaging system.

[0039] Results analysis: ImageJ software was used to analyze the grayscale values ​​of the bands and compare the differences between the groups.

[0040] The results are as follows Figure 1 As shown in Figure A, the sodium lactate treatment group with a Klac / actin ratio of 20 mM > the sodium lactate treatment group with a Klac / actin ratio of 10 mM > the blank control group, demonstrating that a high lactate environment enhances the lactation modification of MSC proteins.

[0041] 4. Osteogenic differentiation induction protocol: (1) When the cells have fused to 70%-80%, the culture medium should be replaced with osteogenic induction medium.

[0042] Osteogenic induction medium formulation: Add 10% FBS, 10 mM β-Glycerophosphate, 50 μM ascorbic acid, 100 nM dexamethasone, and 1% penicillin-streptomycin to the basal medium (α-MEM).

[0043] Induction period: Replace with fresh osteogenic induction medium every 2-3 days and continue culturing for 14 days.

[0044] Experimental groups: Control group: normal osteogenic induction medium; High sodium lactate group (20 mM Na-lac): osteogenic induction medium + 20 mM sodium lactate; Low sodium lactate group (10 mM Na-lac): osteogenic induction medium + 10 mM sodium lactate.

[0045] (2) On day 14 after induction, Alizarin Red S (ARS) staining was used to detect calcium nodule formation: Fixation: Discard the culture medium, wash twice with PBS, and fix with 4% PFA at room temperature for 30 minutes.

[0046] Staining: Discard the fixative and wash twice with PBS. Add an appropriate amount of 2% ARS staining solution (pH 4.1-4.3) and incubate at room temperature in the dark for 20-30 minutes.

[0047] Cleaning and photography: Discard the staining solution and gently wash 4-5 times with ultrapure water until the eluent is colorless to remove non-specific staining. Take a whole-well photograph under a microscope to see orange-red mineralized nodules.

[0048] Quantitative analysis: For semi-quantitative analysis, 10% cetylpyridine chloride (CPC) solution was added to the stained wells and incubated with shaking at room temperature for 30 minutes to dissolve the bound dye. The solution was then pipetted and its absorbance was measured at 562 nm.

[0049] The results are as follows Figure 1 As shown in Figure B, the number of mineralized nodules and the absorbance value at 562 nm were both: high lactate group < low lactate group < control group, demonstrating that lactation modification inhibits the osteogenic function of MSCs.

[0050] Example 2 To accurately identify protein-specific lactation modification sites that play a key role in inflammatory osteoporosis (AS), this invention employs lactomics, with the specific experimental methods as follows: 1. Sample preparation and peptide preparation: (1) Cell treatment and protein extraction: Fourth-generation human bone marrow MSCs in logarithmic growth phase were collected and divided into two groups: the experimental group was treated with medium containing 20 mM sodium lactate for 48 hours, and the control group was treated with normal medium. After treatment, the cells were washed twice with pre-cooled PBS, scraped off, and lysed with 4 times the volume of lysis buffer (8 M urea, 1% protease inhibitor, 3 μM TSA, 50 mM NAM). The cells were centrifuged at 12000 g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

[0051] (2) Trypsin hydrolysis: Equal amounts of protein from each sample were hydrolyzed, and the volume was adjusted to be consistent with the lysis buffer. 20% TCA was slowly added to a final concentration, vortexed, and precipitated at 4°C for 2 hours. The precipitate was centrifuged at 4500g for 5 minutes, the supernatant was discarded, and the precipitate was washed 2-3 times with pre-cooled acetone. After drying the precipitate, TEAB to a final concentration of 200 mM was added, and the precipitate was sonicated to disperse it. Trypsin was added at a ratio of 1:50 (protease: protein, m / m), and hydrolyzed overnight. Dithiothreitol (DTT) was added to a final concentration of 5 mM, and the mixture was reduced at 56°C for 30 minutes. Iodoacetamide (IAA) was then added to a final concentration of 11 mM, and the mixture was incubated at room temperature in the dark for 15 minutes.

[0052] 2. Enrichment of lactated peptides: The peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0), and the supernatant was transferred to pre-washed lactated resin (antibody resin catalog number PTM-1404, from Hangzhou Jingjie Biotechnology Co., Ltd., PTM Bio). The resin was incubated overnight at 4°C with gentle shaking. After incubation, the resin was washed four times with IP buffer and twice with deionized water. Finally, the resin-bound peptides were eluted three times with 0.1% trifluoroacetic acid eluent. The eluent was collected and freeze-dried under vacuum. After drying, the resin was desalted according to the C18 ZipTips instructions, freeze-dried, and then used for LC-MS / MS analysis.

[0053] 3. Liquid chromatography-mass spectrometry analysis: Peptides were dissolved in mobile phase A of liquid chromatography and then separated using a NanoElute ultra-high performance liquid chromatography (UHPLC) system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was a solution containing 0.1% formic acid and 100% acetonitrile. The liquid phase gradient settings were: 0-42 min, 7%–24% B; 42–54 min, 24%–32% B; 54–57 min, 32%–80% B; 57–60 min, 80% B, with the flow rate maintained at 450.00 nL / min. After separation by the UHPLC system, the peptides were injected into a Capillary ion source for ionization and then analyzed by tims-TOF Pro mass spectrometry. The ion source voltage was set to 2.0 kV, and the precursor ion and secondary fragments of the peptides were detected and analyzed using high-resolution TOF. The secondary mass spectrometry scan range was set to 100–1700. The data acquisition mode used was Parallel Accumulation Serial Fragmentation (PASEF). After a primary mass spectrometer acquisition, 10 secondary spectra were acquired in PASEF mode for precursor ion charges in the range of 0-5. The dynamic exclusion time for tandem mass spectrometry was set to 30 s to avoid duplicate scanning of the precursor ion. 4. Bioinformatics analysis and site identification: Secondary mass spectrometry data were searched using Maxquant 1.6.6.0. Search parameters were set as follows: the database was Homo_sapiens_9606 (20366 sequences), a reverse library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to eliminate the influence of contaminating proteins in the identification results; the restriction enzyme digestion method was set to Trypsin / P; the number of missed cleavage sites was set to 4; the minimum peptide length was set to 7 amino acid residues; the maximum number of peptide modifications was set to 5; the mass error tolerance for primary precursor ions in the First search and Main search was set to 20.0 ppm and 20 ppm, respectively, and the mass error tolerance for secondary fragment ions was 20.0 ppm. Cysteine ​​alkylation (Carbamidomethyl(C)) was set as a fixed modification, with variable modifications ['Acetyl (Protein N-term)', 'Oxidation (M)', 'Lact(K)']. The quantification method was set to LFQ, and the FDR for both protein identification and PSM identification was set to 1%.

[0054] Bioinformatics analysis results ( Figure 2 The results clearly showed that, compared with the control group, the lactation modification level of the lysine 219 (K219) site of the VCL protein in the MSCs of the high lactate treatment group was significantly upregulated, and its modification intensity ranked among the top of all differentially modified sites, indicating that this site is a key pathological modification site in the high lactate microenvironment.

[0055] Example 3 Construction of VCL-K219R overexpressing AAV virus 1. Gene sequence design: Based on the human VCL gene sequence (NCBI Gene ID: 7414), the codon (AAG) of lysine (K) at position 219 was mutated to the codon (AGG) of arginine (R) by site-directed mutagenesis to construct the VCL-K219R mutant. The amino acid sequence is shown in SEQ ID NO: 1. After the whole gene was synthesized, it was cloned into the AAV expression vector.

[0056] 2. Vector construction: VCL-K219R was expressed using the pAAV-MCS vector under the control of an MSC-specific promoter (such as Prx1 or Sca-1 promoter). Simultaneously, AAV vectors expressing wild-type VCL (VCL-WT) and empty vectors (EV) were constructed as controls.

[0057] 3. Virus Packaging and Purification: Recombinant AAV9 virus (serotype 9 exhibits good bone marrow targeting) was packaged in HEK293T cells using a three-plasmid co-transfection method. The virus was purified by iodixanol gradient centrifugation and dialyzed with PBS. Pricing was performed using qPCR, and the final viral titer was adjusted to 1×10⁻⁶. 12 -5×10 12 vg / mL, aliquoted and stored at -80℃.

[0058] Example 4: Application of VCL-K219R overexpression of AAV9 virus in the treatment of inflammatory osteoporosis in AS model mice 1.1 Experimental Materials: (1) Experimental animals: 50 eight-week-old female SKG mice (AS model mice) and 10 wild-type Balb / c mice were provided by the Experimental Animal Center of Sun Yat-sen University.

[0059] (2) Virus: pAAV-VCL-K219R-EGFP (AAV-K219R) and pAAV-EGFP (empty vector control, AAV-EV) prepared according to the above method, both with a titer of 3.5 × 10⁻⁶. 12 vg / mL.

[0060] (3) Reagents: Sodium lactate, osteogenic induction reagent (Sigma), anti-lactation modification antibody (PTM-1401RM), anti-VCL antibody (ab18058), HE and Masson staining kits.

[0061] 2. Experimental methods: (1) Virus injection and grouping: SKG mice were randomly divided into 3 groups (n=15): AS model group (SKG): 100 μL PBS was injected into the tail vein; Empty vector treatment group (SKG+AAV-EV): 100 μL AAV-EV virus solution (containing 3.5×10⁻⁶ PBS) was injected into the tail vein. 11 vg virus); mutant treatment group (SKG+AAV-K219R): 100 μL of AAV-K219R virus solution (containing 3.5×10) was injected via tail vein. 11 (vg virus); another 10 Balb / c mice were used as the normal control group (WT) and injected with an equal amount of PBS.

[0062] (2) Disease induction and observation: One week after viral injection, all SKG mice were intraperitoneally injected with 3 mg of Curdlan suspension to induce arthritis and osteoporosis. The arthritis scores and weight changes of the mice were observed and recorded weekly.

[0063] (3) Sample collection: 12 weeks after viral injection, all mice were anesthetized and femoral and lumbar vertebrae (L4-L6) specimens were collected.

[0064] (4) Bone microstructure assessment (Micro-CT): Micro-CT scan of the distal femur.

[0065] Western blot analysis of proteins extracted from femoral bone marrow cells showed that lactation modification signals of VCL protein were weakened in MSCs from SKG+AAV-K219R group mice (see below) Figure 3 A) demonstrates that VCL-K219R was successfully overexpressed and effectively competitively inhibited the lactation modification of endogenous VCL.

[0066] Quantitative analysis showed that ( Figure 3 (B) Bone mineral density (BMD) and trabecular bone number (Tb.N) were significantly lower in the SKG group than in the WT group, confirming the successful establishment of the osteoporosis model. There was no significant difference between the SKG+AAV-EV group and the SKG group, indicating that the empty vector virus itself has no therapeutic effect. The BMD and Tb.N of the SKG+AAV-K219R group were significantly higher than those of the SKG and SKG+AAV-EV groups, indicating that overexpression of VCL-K219R can effectively reverse bone loss in SKG mice. As shown above, tail vein injection of AAV9-VCL-K219R effectively transfected MSCs in AS model mice, successfully overexpressing the VCL-K219R mutant protein and significantly inhibiting its lactation modification. This treatment effectively improved bone microstructure, enhanced osteogenic activity and bone strength in AS mice, ultimately reversing the progression of inflammatory osteoporosis, and no significant toxic effects were observed at the dose used in this study. This provides solid preclinical evidence for the application of AAV-mediated VCL-K219R gene therapy for AS inflammatory osteoporosis.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A focal adhesion protein mutant, characterized in that, The mutant includes a protein obtained by mutating the amino acid at position 219 of the adhesion protein from lysine to arginine.

2. A nucleotide sequence encoding the adhesion plaque protein mutant of claim 1.

3. An adeno-associated virus, characterized in that, Including the adhesion plaque protein mutant of claim 1.

4. The method of constructing an adeno-associated virus according to claim 3, wherein, The plaque protein mutant described in claim 1 was cloned into a plasmid vector, expressed under the control of an MSC-specific promoter, and transfected into 293T cells. The virus was purified to obtain adeno-associated virus VCL-K219R AAV.

5. A recombinant gene therapy vector, characterized in that, Includes the nucleotide sequence described in claim 2.

6. The use of the focal adhesion protein mutant of claim 1, the adeno-associated virus of claim 3, or the recombinant gene therapy vector of claim 5 in the preparation of medicaments for the prevention and / or treatment of osteoporosis.

7. Use according to claim 6, characterized in that, The osteoporosis mentioned is inflammatory osteoporosis caused by ankylosing spondylitis.

8. The application according to claim 6, characterized in that, The drug promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

9. A pharmaceutical composition for the prevention and / or treatment of ankylosing spondylitis and inflammatory osteoporosis, characterized in that, The pharmaceutical composition comprises the focal adhesion protein mutant of claim 1, the adeno-associated virus of claim 3, or the recombinant gene therapy vector of claim 5.

10. The pharmaceutical composition according to claim 9, characterized in that, It also includes excipients or pharmaceutically acceptable carriers.

Images