Method of making bones and cartilages

Abstract

Described herein are methods of producing cartilage or bone by controlling the expression level of MAST4 in a cell.

Description

Background of the Invention 1. Field of the invention:

[0002] The present invention relates to therapeutic compositions for producing cartilage or bone based on the regulation of MAST4 protein.

[0003] 2. General background and prior art:

[0004] Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various lineages of mesenchymal cell types, including chondrocytes, osteoblasts, and adipocytes. 1 MSC commitment and differentiation into each individual cell type depends on multiple signaling pathways, including Wnt, TGF-β, BMP, and FGF 2 During differentiation, the coordinated upregulation and repression of transcription factors are triggered through specific signaling pathways and interactions with many other transcription factors that act as co-regulators.

[0005] Sox9, a member of the high-mobility group (HMG) domain transcription factor family, is an activator of chondrogenesis and regulates the process from the initiation of pre-cartilaginous condensation to the terminal differentiation of chondrocytes. 3-5 Sox9 activates collagen genes (Col2, Col9, Col11) and cartilage matrix genes (Acan and Comp) by directly binding to their enhancers and promoters. 6,7 Considering that Sox9 is a key regulator of chondrogenesis, Sox9 is strictly regulated by multiple mechanisms. 8 Several studies have reported phosphorylation events that regulate Sox9 in chondrocytes. 9-11 .

[0006] TGF-β signaling is involved in cartilage development and maintenance, especially in stimulating chondrocyte differentiation during the early stages of chondrogenesis 12,13 Animal studies have shown that Smad3 is a key mediator of TGF-β1 signaling and is required for the maintenance of articular cartilage, and that mice with Smad3 deficiency or chondrocyte-specific depletion of Smad3 result in degeneration of articular cartilage. 14,15 In addition, previous studies have shown that TGF-β1 signaling promotes chondrogenesis by regulating Sox9 in a Smad3-dependent and -independent manner. 16-18 , which means that the TGF-β1-Sox9 axis is crucial in regulating chondrogenesis.

[0007] Wnt / β-catenin signaling plays a crucial role in endochondral ossification by regulating osteoblast differentiation and maturation 19 Wnt-induced intracellular stabilization of β-catenin and subsequent nuclear translocation lead to the activation of Runx2, a major transcription factor for osteoblast differentiation, specifically in mesenchymal cells that develop into bone. 20 In addition, GSK-3β, a key negative regulator of canonical Wnt / β-catenin signaling, has been shown to reduce Runx2 activity during osteogenesis, suggesting that GSK-3β is a potential molecular target for the treatment of bone diseases. 21 .

[0008] In this work, considering that protein kinases play a vital role in signal transduction, we have sought to identify genes that may be involved in the regulation of switching mesenchymal progenitor cells to a specific lineage downstream of TGF-β or Wnt signals. In this article, we found that microtubule-associated serine / threonine kinase 4 (Mast4) plays a vital role in determining the cell fate of MSCs to become chondrocytes or osteoblasts, and the microtubule-associated serine / threonine kinase 4 (Mast4) is inhibited by TGF-β1 during chondrogenesis of MSCs and enhanced by Wnt-mediated GSK-3β inhibition during osteogenesis of MSCs. We show that Mast4-induced Sox9 phosphorylation at serine 494 residues leads to proteasomal degradation of Sox9. We further demonstrate that Mast4 deficiency leads to increased Sox9 stability and Smad3-Sox9 association, resulting in increased transcriptional activity of Sox9 and subsequent expression of chondrocyte marker genes, thereby ultimately promoting chondrogenic differentiation of MSCs. On the other hand, we found that GSK-3β-induced Mast4 phosphorylation triggered Mast4 recruitment of the E3 ligase Smurf1, leading to Mast4 degradation. We then found that Mast4 stabilized by Wnt-mediated GSK-3β inhibition promoted β-catenin nuclear localization, which ultimately increased Runx2 transcriptional activity and subsequent MSC osteogenic differentiation. The effect of Mast4 on chondrogenesis of mesenchymal progenitor cells was confirmed in vivo by showing that Mast4 - / - Excessive cartilage synthesis but osteoporotic bone formation or reduced bone formation in mice was demonstrated. Interestingly, Mast4 depletion in MSCs promoted chondrogenesis and regeneration in vivo. In conclusion, our results reveal an important role for Mast4 in determining the fate of MSCs to develop into cartilage or bone. Summary of the invention

[0009] The present invention relates to a method for manipulating MAST4 expression in cells so that the final product results in the production of cartilage or bone. For example, if the cells are manipulated so that MAST4 is inhibited, the resulting cells will produce additional cell matrix material, and if the cells are administered to a subject's site of interest, cartilage will be produced. Conversely, if the cells are manipulated so that MAST4 is highly expressed, and such cells are administered to a subject's site of interest, bone will be produced.

[0010] In one aspect, the present invention relates to a method for producing bone, comprising administering eukaryotic cells to a subject in need thereof at or near a bone defect site where bone formation is desired, wherein the expression or activity of a microtubule-associated serine / threonine kinase family member 4 (MAST4) protein or a fragment thereof is stabilized or increased relative to normal cells. The method comprises recombinantly expressing MAST4 in the cell.

[0011] The cell can be a connective tissue cell. The eukaryotic cell can be a mesenchymal stem cell, a fibroblast, an osteoprogenitor cell, an osteocyte, a preosteoblast, an osteoblast or an osteoclast. Relative to the host, the eukaryotic cell can be allogeneic or autologous. The cell can recombinantly overexpress MAST4 in the cell. The expressed MAST4 can be controlled by a viral promoter. The viral promoter can be from a lentivirus or an adeno-associated virus.

[0012] In another aspect of the present invention, a cell may be contacted with a composition comprising: (1) a compound that specifically binds to a nucleic acid encoding a MAST4 inhibitory protein, thereby inhibiting the expression of the MAST4 inhibitory protein; or (2) a compound that specifically binds to a MAST4 inhibitory protein, thereby preventing it from binding to MAST4. Without limitation, the MAST4 inhibitory protein may be GSK-3. The inhibitory compound may be a chemical substance, a polypeptide or a polynucleotide, or a combination thereof. The polypeptide may be an antibody or an antigen binding molecule. The inhibitory compound of GSK-3α or GSK-3β may be a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi interacting RNA (piRNA), a small nuclear RNA (snRNA) or an antisense oligonucleotide, or a combination thereof. The compound may also be CRISPR-Cas, which comprises a guide RNA that is specific to a nucleic acid encoding a MAST4 inhibitory protein (or a fragment thereof). The guide RNA can be a double RNA and a single-stranded guide RNA, the double RNA comprising CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), the double RNA is specific to the nucleic acid encoding the MAST4 inhibitory protein (or its fragment), the single-stranded guide RNA comprises a portion of crRNA and tracrRNA and hybridizes with the nucleic acid encoding the MAST4 inhibitory protein (or its fragment). The cell can be a connective tissue cell. The cell can be a mesenchymal stem cell, a fibroblast, an osteoprogenitor cell, an osteocyte, a preosteoblast, an osteoblast or an osteoclast. Relative to the host, the cell can be autologous or allogeneic. The cell can also include a recombinant construct expressing MAST4. The recombinant construct can overexpress MAST4.

[0013] In another aspect, the present invention relates to a method for producing an extracellular matrix from a eukaryotic cell, comprising contacting the eukaryotic cell with a composition comprising a compound capable of specifically binding to a nucleic acid encoding a microtubule-associated serine / threonine kinase family member 4 (MAST4) protein or a fragment thereof and inhibiting the expression or activity of the MAST4 protein, wherein the compound is capable of specifically binding to a nucleic acid encoding the MAST4 protein or a fragment thereof, wherein the eukaryotic cell is a chondrocyte, a fibroblast or a mesenchymal stem cell. The inhibitory compound is a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi interacting RNA (piRNA), a small nuclear RNA (snRNA) or an antisense oligonucleotide, or a combination thereof.

[0014] On the other hand, the present invention relates to a method for preventing, treating or ameliorating joint diseases, the method comprising: (i) administering a compound in eukaryotic cells to inhibit microtubule-associated serine / threonine kinase family member 4 (MAST4) thereby inhibiting the expression or activity of the MAST4 protein; and (ii) administering the eukaryotic cells obtained thereby to a subject in need thereof at or near a joint where cartilage formation is required.

[0015] These and other objects of the present invention will be more fully understood from the following description of the invention, the reference drawings appended hereto, and the claims appended hereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0017] A more complete understanding of the present invention will be obtained from the detailed description given below, together with the accompanying drawings which are given by way of illustration only and therefore not as a limitation of the present invention, and in which:

[0018] Figure 1a-1f shows that targeted deletion of the Mast4 gene enhances the expression of cartilage matrix genes and reduces the expression of in vitro osteogenic genes. a Representative RT-PCR results of high-density micromass culture of C3H10T1 / 2 cells obtained from at least three independent experiments in the presence of BMP-2. b Representative qRT-PCR results of chondrocyte marker gene expression in differentiated wild-type and Mast4-depleted C3H10T1 / 2 cells. During chondrogenic differentiation, protein immunoblotting confirmed the expression of Mast4 protein. c Heat map of DEGs of chondrogenically differentiated wild-type and Mast4-depleted C3H10T1 / 2 cells for 6 days classified according to cartilage / chondrocyte development and osteogenesis. d Representative qRT-PCR results of Sox9 targeted genes and Mmp9 / 13 identified by RNA sequencing in wild-type and Mast4-depleted C3H10T1 / 2 cells differentiated into chondrocytes for 6 days. e Representative qRT-PCR results of osteoblast marker genes and osteogenesis-related genes identified in c in wild-type and Mast4-depleted C3H10T1 / 2 cells differentiated into osteoblasts for 10 days. f Transcriptional network of DEGs related to cartilage and chondrocyte development, TGF-β signaling, and BMP signaling in wild-type and Mast4-depleted C3H10T1 / 2 cells differentiated into chondrocytes for 6 days. Node size was set based on the connectivity of the nodes. Red circles indicate genes showing differential expression in Mast4-depleted C3H10T1 / 2 cells. b, d, and e data are representative means ± SD of three independent experiments, each repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing. P values ​​are compared with WT on the corresponding day.

[0019] Figure 2 a-2l shows that Mast4 regulates chondrogenesis through post-translational regulation of Sox9. a Alcian blue staining results of C3H10T1 / 2 cells. b 2×10 5hBMSCs were differentiated into chondrocytes for 21 days, and then proteins were extracted from the pellets. c C3H10T1 / 2 cells were differentiated into chondrocytes for 6 days, and then Sox9 ChIP was performed on the Col2a1 gene (TGF-β1 (5 ng / ml) and TGF-β type I receptor kinase inhibitor Vactosertib (0.5 μM) for 48 h). d 4xCol2a1-luc, Sox9, and Mast4-PDZ were transiently overexpressed in wild-type and Mast4-depleted C3H10T1 / 2 cells, followed by TGF-β1 treatment (3 ng / ml for 24 h). e Mast4-PDZ and Sox9 were transfected into C3H10T1 / 2 cells, and then immunoprecipitation assays were performed. f Western blot analysis of Sox9 stability in C3H10T1 / 2 cells. g Wild-type and Mast4-depleted C3H10T1 / 2 cells were differentiated into chondrocytes for 6 days, followed by TGF-β1 (5 ng / ml for 48 h) and MG-132 (10 μM for 6 h) treatment, and Sox9 immunoprecipitation. Band intensities representing pSerine and Sox9 were converted by densitometry using ImageJ. Data are reported as mean ± SD of three independent experiments (n = 3). Statistical analysis was performed using an unpaired two-tailed Student's t-test (P < 0.05). h Sox9 and Mast4-PDZ were transfected into C3H10T1 / 2 cells in the presence of MG-132 (10 μM for 6 h). Two independent MASS SPEC analyses were performed. i Mast-4PDZ was co-transfected with Sox9 wild-type (WT), S494A, or S494D mutant into C3H10T1 / 2 cells. j 4xCol2a1-luc and Sox9WT / S494A / S494D were co-transfected into C3H10T1 / 2 cells, followed by TGF-β1 treatment (3 ng / ml for 24 h). k Representative Alcian blue staining of C3H10T1 / 2 cells stably overexpressing Sox9WT, S494A and S494D for 6 days in micromass culture. l Wild-type and stably overexpressing Sox9WT, S494A, S494D C3H10T1 / 2 cells were differentiated into chondrocytes for 6 days. mRNA expression was detected by RT-PCR. c, d and j data are representative mean ± SD of three independent experiments, each repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing. a, b, eg, i and l representative results were obtained from at least three independent experiments. TCL: total cell lysate.

[0020] Figure 3a-3g shows that TGF-β1-induced Mast4 inhibition enhances chondrogenesis by increasing Sox9-Smad3 association. a Sox9 and Smad3 were co-transfected into wild-type and Mast4-depleted C3H10T1 / 2 cells, followed by TGF-β1 treatment (5 ng / ml for 30 min). Sox9 was immunoprecipitated using HA antibody. The band intensities representing the expression levels of GFP-Smad3 and HA-Sox9 in the immunoprecipitates were converted to the ratio of Smad3 to Sox9 by densitometry using ImageJ software. Data are reported as the mean ± SD of three independent experiments (n = 3). Statistical analysis was performed using an unpaired two-tailed Student's t-test (P < 0.05). b Smad3 ChIP gene detection on Col2a1 was performed in differentiated C3H10T1 / 2 cells for 6 days. c Mast4 expression was detected by Western immunoblotting and RT-PCR in C3H10T1 / 2 cells treated with TGF-β1 for 48 h. d Luciferase assay in undifferentiated C3H10T1 / 2 cells. Vactosertib (0.5 μM) pretreatment lasted for 2 h, followed by TGF-β1 (5 ng / ml) treatment for 24 h. e Smad3ChIP detection of Mast4 gene in C3H10T1 / 2 cells undergoing chondrogenic differentiation for 6 days. TGF-β1 (5 ng / ml) and Vactosertib (0.5 μM) were treated for 48 h and 50 h, respectively, before harvesting. f Endogenous Mast4 and Sox9 protein expression was detected in differentiated C3H10T1 / 2 cells by Western blotting. g TGF-β1 (5 ng / ml) was treated for 24 h and 48 h, and Vactosertib (0.5 μM) was treated for 48 h to human primary chondrocytes. b, d, and e Data are representative means ± SD of three independent experiments, each repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) with Benjamini-Hochberg correction for multiple testing. a, c, f, and g are representative results obtained from at least three independent experiments. TCL: total cell lysate.

[0021] Figure 4a-4l shows that Wnt enhances the stability of Mast4 by inhibiting GSK-3β, thereby inducing osteogenesis. a Representative ALP staining results of osteogenic differentiated C3H10T1 / 2 cells obtained from at least three independent experiments. b MC3T3-E1 preosteoblasts were treated with Wnt3a-conditioned medium for the indicated time. C Wild-type and Mast4-PDZ-overexpressing C3H10T1 / 2 cells were differentiated into osteoblasts for 10 days. d Mast4-PDZ and GSK-3β were transfected into C3H10T1 / 2 cells, followed by CHIR-99021 treatment (10 μM for 9 h). e Mast4-PDZ and GSK-3β were transfected into C3H10T1 / 2 cells, followed by immunoprecipitation assay. The bands were recognized by phospho-serine antibody, and then the bands were reprobed with HA antibody. f Smurf1, Mast4-PDZ and GSK-3β were transfected into C3H10T / 12 cells treated with CHIR-99021 (10 μM for 9 h), followed by immunoprecipitation assay. g Smurf1 and GSK-3β were transfected into wild-type and GSK-3β-depleted Mast4-PDZ-overexpressing C3H10T1 / 2 cells. h Mast4-PDZ WT, P628A / Y634A and Smurf1 were transfected into wild-type and GSK-3β-depleted C3H10T1 / 2 cells. i Each Mast4 kinase domain deletion mutant and GSK-3β were transfected into C3H10T1 / 2 cells. j Flag-Mast4-PDZ WT and Δ632-636 were transfected into C3H10T1 / 2 cells with HA-ubiquitin in the absence or presence of Smurf1 and GSK-3β. Cell lysates were immunoprecipitated using Flag antibodies and immunoblotted using designated antibodies. K MC3T3-E1 cells were transfected with Mast4-PDZ WT and Δ632-636, followed by treatment with Wnt3a conditioned medium (2.5 h). l 6xOSE-Luc, Mast4-PDZ WT and Δ632-636 were transfected into C3H10T1 / 2 cells, followed by treatment with Wnt3a conditioned medium (18 h). Data are representative mean ± SD of three independent experiments, each experiment was repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing. bk: representative results obtained from at least three independent experiments. TCL: total cell lysate.

[0022] Figure 5 a-5h shows that Mast4 depletion induces changes in chondrogenesis and osteogenesis during development. a, b Mast4+ / + and Mast4 - / - Immunofluorescence images of Sox9 and Col2a1 in the growth plates of mice (n=3). c, d (c) 1-day-old and (d) 3-week-old Mast4 + / + and Mast4 - / - Five-color staining of mouse tibial growth plate (n=5). Black arrows indicate the hypertrophic zone. e Mast4 + / + and Mast4 - / - μCT images of trabecular bone in the tibia of mice. Total bone volume and bone mineral density (BMD) of trabecular bone were measured from μCT images (Mast4 + / + Mouse: n=8, Mast4 - / - f Bone formation was observed by calcein double labeling at 7 and 2 days before sacrifice, and the distance between labels was measured at 3 points on each tibia (Mast4 + / + n = 10 tibiae and Mast4 - / - MAR: mineral apposition rate. g 6-week-old Mast4 + / + and Mast4 - / - Representative images of immunofluorescence staining of Osterix and Mmp13 near metaphyseal vessels (CD31) in proximal tibiae of mice (n=3). GP: growth plate, MP: metaphysis. h Using 5-week-old Mast4 + / + and Mast4 - / - Representative alcian blue and alizarin red S staining results obtained from in vitro colony formation assays of skeletal stem cells isolated from mice (n=5). e, f, Data are reported as mean ± SD. All statistical analyses were performed using unpaired two-tailed Student's t-test (P<0.05).

[0023] Figure 6 a-6e show the identification of genes regulated by Mast4 in mouse cartilage and bone. a, b were obtained by collecting and merging the genes regulated by Mast4 in mouse cartilage and bone at 1 day PN. + / + and Mast4 - / - RNA sequencing was performed on RNA obtained from the cartilage and bones of the tibia of mice (3 mice per group). a Mast4 - / - Enrichment of up-regulated genes related to cartilage development in mouse cartilage. 20 up-regulated genes were predicted as the leading-edge subset of the enriched gene set. b Mast4+ / + Enrichment of upregulated genes related to skeletal system development and Wnt signaling pathway in mouse bones. In the plot, 20 and 12 upregulated genes were included as + / + A leading subset of enriched gene sets in mouse. Gene set enrichment analysis was performed using a background dataset generated by Mast4 + / + and Mast4 - / - All DEGs analyzed in tibial cartilage and bone of mice. c Transcriptional network of Sox9 and Runx2 targets regulated by Mast4 showing differential expression in cartilage and bone. Sox9 and Runx2 targets are associated with genes related to skeletal system developmental functions, including cartilage and bone development, TGF-β signaling, BMP signaling, and Wnt signaling. d Mast4 at PN 1 day + / + and Mast4 - / - Western blot analysis of Col2a1 and Sox9 proteins in cartilage tissue of mice. e Mast4 at 1 day PN + / + and Mast4 - / - Western blot analysis of Runx2, Mmp13, and β-catenin in cartilage tissue of mice. d, e Representative results were obtained from at least three independent experiments.

[0024] Figure 7a-7d shows that Mast4-depleted MSC transplantation improves chondrogenesis and repair in vivo. a 100 micromass cultures of wild-type and Mast4-depleted C3H10T1 / 2 cells cultured in 150 mm dishes with chondrogenic differentiation medium were subcutaneously injected into each side of athymic nude mice (n=4; WT on the left and KO on the right). Ectopic masses were retrieved 2 weeks after implantation. The values ​​given are the mean ± SD of the volume of the grafts from 4 mice. All statistical analyses of (a) were performed using an unpaired two-tailed Student's t-test (P<0.05). b Multiple cartilage structures found in wild-type and Mast4-depleted C3H10T1 / 2 cell transplants were confirmed by immunofluorescence staining for Col2a1 and Sox9. The incorporation of Sox9 and TOPRO-3 is shown in yellow. Representative images were obtained from immunostaining of 1 cartilage nodule formed by wild-type C3H10T1 / 2 cells and 7 different cartilage nodules formed by Mast4-depleted C3H10T1 / 2 cells. c Macroscopic and microscopic appearance of full-thickness cartilage defects in the trochlear groove in the rabbit model at 12 weeks after transplantation. Vehicle: PBS treatment without hBMSC transplantation. d Wakitani cartilage repair score of regenerated cartilage. Data are expressed as mean ± SD. c, d Vehicle-treated group n = 3, original (naive) group and MAST4-depleted hBMSC transplantation group n = 5. Statistical analysis of (d) was performed using one-way ANOVA with Dunnett correction for multiple comparisons.

[0025] Figure 8 Gene expression during chondrogenesis of ATDC5 cells is shown. ATDC5 cells were treated with insulin 100 ng / ml for 6 and 9 days to induce chondrogenesis, and RT-PCR was performed. Representative results were obtained from at least three separate experiments.

[0026] Fig. 9a-9e shows the effect of Mast4 depletion on mRNA expression of chondrocyte marker genes. (a) Analysis of CRISPR / Cas9-mediated deletion of Mast4 exon 1 and exon 2 in C3H10T1 / 2 cells. Exon 1 and exon 2 of the Mast4 locus were amplified to distinguish genotypes, and PCR products were used for sequencing. Two bases were deleted in both chains of exon 1, inducing nonsense mutations in exon 1 and translation termination. Seven base deletions and one base insertion were found in exon 2, inducing biallelic mutations in the exon and translation termination. (b) Depletion of Mast4 protein was confirmed by protein immunoblotting using undifferentiated cells. (c and d) Expression of chondrocyte marker genes in undifferentiated (c) wild-type and Mast4 depleted, (d) control and shRNA-mediated Mast4 knockdown C3H10T1 / 2 cells. (d) Data are representative means ± SD of three independent experiments, each repeated three times (n = 3). Statistical analysis was performed using an unpaired two-tailed Student's t-test (P < 0.05) with Benjamini-Hochberg correction for multiple testing. P values ​​are compared with WT. (e) Expression of chondrocyte marker genes was detected in wild-type and Mast4-depleted C3H10T1 / 2 cells induced to chondrogenic differentiation for 6 days. (b, c, and e) Representative results obtained from at least three separate experiments.

[0027] Fig.10 a-10b shows Sox9 expression in wild-type and Mast4-depleted C3H10T1 / 2 cells. (a) Endogenous Sox9 protein expression was detected in undifferentiated wild-type and Mast4-depleted C3H10T1 / 2 cells. The band intensity representing the Sox9 expression level was converted to the ratio of Sox9 to b-actin by densitometry using ImageJ software. Representative results were obtained from at least three separate experiments. (b) The mRNA expression of Sox9 in undifferentiated wild-type and Mast4-depleted C3H10T1 / 2 cells was detected by qRT-PCR. The data are representative means ± SD of three independent experiments, each experiment was repeated three times (n = 3). All statistical analyses were performed using an unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing.

[0028] Fig.11a-11b shows forced expression of truncated Mast4 protein in C3H10T1 / 2 cells. (a) Schematic structure of full-length and truncated Mast4 constructs. (b) RT-PCR was performed using control (LPCX) and Mast4-PDZ overexpressing C3H10T1 / 2 cells to analyze mRNA expression of chondrocyte marker genes. Representative results were obtained from at least three separate experiments.

[0029] Fig.12 DEG and GO enrichment analysis of WT and Mast4-depleted C3H10T1 / 2 cells are shown. Wild-type and Mast4-depleted (KO#1) C3H10T1 / 2 cells were differentiated into chondrocytes for 6 days under BMP-2 stimulation in high-density micromass culture and then subjected to RNA sequencing analysis. Differentially expressed gene (DEG) analysis and gene ontology (GO) enrichment analysis were performed.

[0030] Fig.13 Shown is the expression of collagen gene family in wild-type and Mast4-depleted C3H10T1 / 2 cells. RNA sequencing shows changes in the expression of collagen gene family in wild-type and Mast4-depleted C3H10T1 / 2 cells.

[0031] Fig.14 a-14e shows the effect of Mast4 depletion in C3H10T1 / 2 cells on chondrogenesis in vitro. (a) Schematic diagram of chondrogenic differentiation of C3H10T1 / 2 cells in a high-density micromass culture system. (b) 5×10 3 Undifferentiated wild-type cells and Mast4-depleted C3H10T1 / 2 cells were seeded in 6-well plates. After chondrogenic differentiation under BMP-2 stimulation, two micromass cultures (1×10 5 / 10ml droplets) were seeded in 12-well plates. The number of cells was counted using a Countess II automated cell counter at the indicated time points. Data are representative means ± SD of three independent experiments, each repeated three times (n = 3). All statistical analyses were performed using an unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing. P values ​​are compared with WT. (c) Representative Alcian blue staining results obtained from at least three independent experiments using chondrogenically differentiated C3H10T1 / 2 cells. (d) After 8 days of chondrogenic differentiation, 1 × 10 5Wild-type and Mast4-depleted C3H10T1 / 2 cells were plated in low-binding 96-well plates. Representative phase contrast microscopy images of 3D spheroids were obtained from at least three independent experiments. (e) mRNA was isolated from the spheroids shown in (d) and the expression of chondrogenic marker genes was analyzed by RT-PCR. (c and e) Representative results were obtained from at least three separate experiments.

[0032] Fig.15 a-15b shows the chondrogenic differentiation of human bone marrow-derived stem cells (hBMSC). (a) Schematic diagram of the chondrogenic differentiation of human bone marrow-derived stem cells (hBMSC) in a pellet culture system. (b) 2×10 5 hBMSCs were differentiated into chondrocytes for 21 days, and then mRNA was isolated from aggregates. The mRNA expression of Col2a1 and Acan was detected by qRT-PCR. Undif.: undifferentiated hBMSCs. Data are representative means ± SD of three independent experiments, each repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing.

[0033] Fig.16a-16e shows that Mast4 regulates Sox9 binding stability to Col2a1 and Sox9. (a) The indicated C3H10T1 / 2 cells were differentiated into chondrocytes for 6 days, and then Sox9 ChIP was performed on the Col2a1 gene. (b) 4xCol2a1-luc was transiently overexpressed in LPCX control and Mast4PDZ-overexpressing C3H10T1 / 2 cells, followed by TGF-b1 treatment (3ng / ml for 24h). (a and b) Data are representative mean ± SD of three independent experiments, each repeated three times (n = 3). All statistical analyses were performed using an unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing. P values ​​are compared with WT. (c) Full-length (HA-Mast4-FULL) and truncated Mast4 (HA-Mast4-PDZ) were co-transfected with siMAST4 (50 pmol) targeting exogenous Mast4 (human) and Myc-Sox9 into C3H10T1 / 2 cells. Since the efficiency of detecting full-length Mast4 using HA antibody was low, a Mast4 antibody recognizing the C-terminus was used. (d) The indicated C3H10T1 / 2 cells were differentiated into chondrocytes for 6 days and then treated with MG-132 (10 mM for 6 h). Endogenous Sox9 was immunoprecipitated and the complex was analyzed by Western blot analysis using phospho-serine and Sox9 antibodies. (e) Myc-Sox9 and Mast4-PDZ constructs were transfected into C3H10T1 / 2 cells and then treated with MG-132 (10 mM for 6 hours). (ce) The band intensities representing the expression levels of Myc-Sox9 or serine-phosphorylated Sox9 were converted to the ratio of Myc-Sox9 or pSerine to b-actin or Sox9 in the immunoprecipitates by densitometry using ImageJ software. Representative results were obtained from at least three separate experiments.

[0034] Fig.17 Mast4 is shown to regulate Sox9 by phosphorylation at serine 494 of Sox9. As described above, 4xCol2a1-luc, Sox9WT / S494A / S494D and Mast4-PDZ were transiently overexpressed in wild-type and Mast4-depleted C3H10T1 / 2 cells. Data are representative mean ± SD of three independent experiments, each experiment was repeated three times (n = 3). Statistical analysis was performed using an unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing.

[0035] Fig.18a-18c shows the interaction of Mast4 with Smad3 and its effect on TGF-b1 / Smad3-induced transcriptional activation. (a) The indicated plasmids were co-transfected into C3H10T1 / 2 cells and then subjected to immunoprecipitation assays. Representative results were obtained from at least three separate experiments. (b and c) Smad3 / 4-induced transcriptional activation was detected in wild-type and Mast4-depleted C3H10T1 / 2 cells by (b) CAGA and (c) SBE reporter gene assays. Data are representative mean ± SD of three independent experiments, each experiment was repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-tests (P < 0.05) and Benjamini-Hochberg correction for multiple testing.

[0036] Fig.19 a-19b shows the effect of Mast4 on Smad3 binding to Smad7 and TGF-b1 target gene expression. (a) Smad3ChIP assay was performed in wild-type and Mast4-depleted differentiated C3H10T1 / 2 cells, which examined the binding of Smad3 to Smad7. Data are representative mean ± SD of three independent experiments, each experiment was repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple tests. (b) mRNA expression of TGF-b1 target genes was examined in wild-type (WT), Mast4-depleted (KO; KO#1) and Mast4-PDZ-overexpressed (OE) C3H10T1 / 2 cells.

[0037] Fig. 20 a-20b shows TGF-b1 regulation of Mast4 promoter activity and Smad3 binding to Mast4 promoter by Smad3ChIP assay. (a) The expected binding sites of Smad3 / 4, p300, c-Jun, Nkx2.1 and E2F-4 transcription factors in Mast4 promoter were predicted using PROMO v3.0.2 software. (b) Smad3ChIP assay was performed on Mast4 gene in differentiated (6 days in chondrogenic differentiation medium) C3H10T1 / 2 cells in the presence or absence of TGF-b1 (5ng / ml for 48h). Data are representative mean ± SD of three independent experiments, each experiment was repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing.

[0038] Fig.21The mRNA expression of Mast4 and chondrocyte marker genes in differentiated C3H10T1 / 2 cells treated with vactosertib is shown. Vactosertib (0.5 mM) was treated for 3 days and 6 days in differentiated C3H10T1 / 2 cells. The mRNA expression of chondrocyte marker gene expression was detected by RT-PCR. Representative results were obtained from at least three separate experiments.

[0039] Fig. 22 a-22e shows the induction of Mast4 during osteogenesis and GSK-3b regulation of Mast4 expression. (a) Schematic diagram of osteogenic differentiation of C3H10T1 / 2 cells. (b) The indicated C3H10T1 / 2 cells were differentiated into osteoblasts for 10 days and then stained with Alizarin Red S. (c) C3H10T1 / 2 cells were differentiated into osteoblasts for 10 days and then subjected to Western blot analysis for the expression of Mast4 and key Wnt signaling molecules. (d) Mast4-PDZ-overexpressing C3H10T1 / 2 cells were treated with cycloheximide (CHX; 10 mg / ml) in the absence or presence of CHIR-99021 for the indicated times (10 mM for 9 h). (e) Wild-type and GSK-3b-depleted HA-Mast4-PDZ-overexpressing C3H10T1 / 2 cells were treated with MG-132 (10 mM for 4 h) and CHIR-99021 (10 mM for 9 h). (bd) Representative results obtained from at least three separate experiments.

[0040] Fig.23 a-23c shows the binding of GSK-3b and Smurf1 to the kinase domain of Mast4. (a) Schematic diagram of Mast4-PDZ deletion mutants. (b) GFP-GSK-3b, (c) GFP-Smurf1 and various deletion mutants of Mast4 were co-transfected into C3H10T1 / 2 cells and then immunoprecipitated. Representative results were obtained from at least three separate experiments.

[0041] Fig.24a-24b shows the enhancement of Runx2 activity and osteogenic differentiation by Mast4-PDZ overexpression and GSK-3b depletion. (a) 6xOSE-Luc was transfected into wild-type and GSK-3b-depleted wild-type and Mast4-PDZ-overexpressing C3H10T / 12 cells, and then treated with Wnt3a-conditioned medium for 18 h. (b) Wild-type and GSK-3b-depleted wild-type and Mast4-PDZ-overexpressing C3H10T / 12 cells were differentiated into osteoblasts for 10 days, and then the mRNA expression of osteoblast marker genes was examined. (a and b) Data are representative means ± SD of three independent experiments, each repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing.

[0042] Fig.25 a-25e shows the analysis of CRISPR / Cas9-mediated deletion of Mast4 exon 1 and exon 15. (a) Delineation of the mouse Mast4 locus using boxes of exons. - / - Allele structure. (bc) Exon 1 and exon 15 of the Mast4 locus were amplified to distinguish the genotypes, and the PCR products were sequenced. As shown in the sequencing results, 71 bases were deleted in exon 1, resulting in nonsense mutation and translation termination of exon 1; at the same time, 3 bases were deleted in exon 15, thereby removing 1 arginine. (d) Western blot analysis in the brain and muscle of young mice confirmed that Mast4 - / - Loss of Mast4 protein in mice. (e) shows the loss of Mast4 in 6-week-old mice. + / + 、Mast4 + / - and Mast4 - / - Representative images of body dimensions of mice. (b and d) Representative results obtained from at least three separate experiments.

[0043] Fig.26 A-26E shows Mast4 + / + and Mast4 - / - Mast4 expression pattern, tibial growth plate thickness and mast cell area ratio in mice. (a) Mast4 + / + and Mast4 - / -Expression pattern of Mast4 in mouse tibia (n=3). The white dotted lines show the boundaries between the proliferating zone, mast cell layer, and ossification zone. P: proliferating zone, H: mast cell layer, O: ossification zone. (b) Pentachrome staining and H&E staining images of tibial growth plate (n=3). (a and b) Expression of Mast4 in mouse tibia + / + and Mast4 - / - Representative images obtained from immunostaining of mice (n=3). (ce) Total growth plate thickness and mast cell layer thickness (Mast4 + / + and Mast4 - / - Mice were treated with PN for 1 day: n = 4, PN for 3 weeks: n = 5, and PN for 6 weeks: n = 3). The ratio of the mast cell layer was calculated as the mast cell layer thickness / growth plate thickness. Data are reported as mean ± SD. Statistical analysis of (c, d, e) was performed using an unpaired two-tailed Student's t-test (P < 0.05). P values ​​are compared with WT.

[0044] Fig. 27 a-27d shows mCT analysis, elemental mapping by electron probe microanalyzer and Mast4 + / + and Mast4 - / - Limb length of mice. (a) Reconstructed 3D mCT image of tibia. From Mast4 + / + 、Mast4 + / - and Mast4 - / - Representative images obtained by immunostaining of mice (n=3). (b) mCT images were used to examine Mast4 in 6-week-old mice. + / + and Mast4 - / - Bone volume, bone density, and cortical bone thickness of the tibia of mice. (c) 6-week-old Mast4 + / + and Mast4 - / - Elemental mapping of calcium (Ca), phosphorus (P), and magnesium (Mg) in the tibia of mice. The red parts indicate high element concentrations, while the dark blue parts indicate low concentrations. BEI: backscattered electron image. (d) Examination of 6-week-old Mast4 using mCT images + / + and Mast4 - / - Limb length of mice. A cross section was set up including the axis passing through the two points farthest from the bone, and the longest length was measured. (b and d) Data are reported as mean ± SD (Mast4 + / + Mice: n = 8 and Mast4 - / - Mice: n = 10). Unpaired two-tailed Student's t-test (P < 0.05) was used for statistical analysis (b, d). P values ​​are compared with WT.

[0045] Fig.28 a-28b shows Mast4 at 6 weeks old + / + and Mast4 - / - Expression of osteoblast marker proteins in the proximal tibia and distal femur of mice. (a) In Mast4 + / + and Mast4 - / - Expression of Runx2 in the proximal tibial metaphysis of mice. GP: growth plate, MP: metaphysis. Images showing the expression of Osterix and Mmp13 correspond to Figure 5 g. (b) Mast4 at 6 weeks of age - / - The expression of Runx2, Osterix and Mmp13 in the periosteum of the distal femur of mice was reduced. P: periosteum, C: cortex, M: medulla. (a and b) From Mast4 + / + and Mast4 - / - Representative images obtained from immunostaining of mice (n=3).

[0046] Fig.29 a-29b shows a sample of Mast4 from 5 weeks old + / + / + and Mast4 - / - Isolation of mouse skeletal stem cells in mice. (a) Representative FACS plots as generated on a BD FACS Aria II and analyzed by FlowJo v10.7.1 and BD FACSDiva v.9.0.1 software, including the percentage of the parental gate for each population. (b) Antibodies and concentrations used to isolate mouse skeletal stem cells.

[0047] Fig.30 A-30C shows the results from Mast4 + / + and Mast4 - / - Functional evaluation of mouse skeletal stem cells isolated from mice. (a) Schematic diagram of in vitro colony formation assay of mouse skeletal stem cells. (b and c) Absorbance measurements were performed by extracting stained colonies using cloning cylinders to distinguish each colony. Alcian blue and Alizarin Red S staining were quantified by absorbance measurements at 630 nm and 405 nm, respectively. Data are reported as the mean ± SD of three stained colonies (n = 3). Statistical analysis of (b, c) was performed using an unpaired two-tailed Student's t-test (P < 0.05).

[0048] Fig.31 a-31d shows the chondrogenic and osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (mBMMSCs). (a) Schematic diagram of mouse bone marrow-derived mesenchymal stem cells (mBMMSCs) differentiated into chondrocytes or osteocytes. (b) + / + and Mast4- / - Mouse BMMSCs were isolated from bone marrow aspirates harvested from the tibia marrow compartment of mice and then induced for chondrogenic differentiation for 21 days and subsequently stained with Alcian blue (n=3). (c) Expression of chondrocyte marker genes in differentiated BMMSCs shown in (b). (d) Expression of chondrocyte marker genes in differentiated BMMSCs from Mast4 + / + and Mast4 - / - mBMMSCs isolated from mice were cultured in osteoblast medium and stained with alkaline phosphatase (ALP). (c and d) Representative results were obtained from at least three separate experiments.

[0049] Fig.32 a-32b shows the identification of genes regulated by Mast4 in mouse cartilage and bone. (a) DEGs were classified into 5 clusters based on changes in gene expression in cartilage and bone. (b) GO enrichment analysis of DEGs within the 5 clusters. A~M represent enriched GO terms (P<0.001). The box plot in (a) shows the enrichment scores of the GO terms described in (b). The grayscale heat map in (a) represents the DEGs associated with the GO terms.

[0050] Fig.33 a-33d show Mast4 regulation of Sox9 target genes and osteogenesis-related genes in mouse cartilage and bone, respectively. + / + and Mast4 - / - (a and c) Heat map and (b and d) qRT-PCR validation of mRNA expression of Sox9 target genes in cartilage tissue of mouse tibia and mRNA expression of osteogenesis-related genes in bone tissue. (b and d) Data are representative mean ± SD of three independent experiments, each experiment was repeated three times (n = 3). All statistical analyses were performed using unpaired two-tailed Student's t-test (P < 0.05) and Benjamini-Hochberg correction for multiple testing.

[0051] Fig.34a-34b shows the effect of Mast4 depletion in C3H10T1 / 2 cells on chondrogenesis in vivo. (a) Schematic diagram of the preparation of differentiated C3H10T1 / 2 cells for subcutaneous injection into the flanks of 4 athymic nude mice (6-week-old females). The mice were sacrificed 2 weeks later and the grafts were retrieved for further analysis. (b) Multiple cartilage structures found in the clumps formed by Mast4-depleted C3H10T1 / 2 cells were confirmed by pentachrome staining. The images in each rectangle are presented at a greater magnification. Scale bar = 100 mm. Representative images were obtained from immunostaining of 1 cartilage nodule formed by wild-type C3H10T1 / 2 cells and 7 different cartilage nodules formed by Mast4-depleted C3H10T1 / 2 cells.

[0052] Fig.35 a-35c shows the transplantation of hBMSCs into full-thickness cartilage defects in a rabbit model. (a) Macroscopic appearance of full-thickness cartilage defects in a rabbit model and microscopic appearance of H&E, Safranin O, Masson's trichrome, and immunohistochemical detection of type II collagen and human HLA class 1 ABC in the defect in the trochlear groove at 12 weeks after transplantation. Vehicle: PBS treatment without hBMSC transplantation. Representative images obtained from vehicle-treated groups n=3, naive groups n=5, and MAST4-depleted hBMSC transplantation groups n=5. (b) The average percentage of collagen area to the entire cartilage defect area evaluated using Masson's trichrome-stained tissue. (c) The average percentage of type II collagen staining area to the entire cartilage defect area. (b and c) Vehicle n=3, naive and CRISPR / Cas9-mediated MAST4KO (pool) human bone marrow-derived stem cells n=5. Data are expressed as mean ± SD. Statistical analysis of (b, c) was performed using one-way analysis of variance (ANOVA) with Dunnett's correction for multiple comparisons.

[0053] Fig.36 a-36b shows the histological grading of regenerated cartilage at the site of full-thickness articular cartilage defect in the rabbit knee joint. (a) Wakitani cartilage repair scoring system. (b) Semi-quantitative analysis of sections using Wakitani scoring. (n=3 for vehicle, n=5 for original and MAST4KO (pool) human bone marrow-derived stem cells). Data are expressed as mean ± SD. Statistical analysis of (b) was performed using one-way analysis of variance (ANOVA) and Dunnett correction for multiple comparisons.

[0054] Fig.37Schematic diagram showing the role of Mast4 in determining the cell fate of MSCs to develop into cartilage or bone. During chondrogenesis, TGF-β1-mediated inhibition of Mast4 leads to an increase in Sox9 protein by reducing Sox9 phosphorylation at S494, resulting in increased Sox9 transcriptional activity, which ultimately causes MSCs to promote chondrogenesis at the expense of bone formation. During osteogenesis, Wnt-mediated inhibition of GSK-3b blocks GSK-3b-induced Mast4 phosphorylation and subsequent Smurf1-mediated degradation of Mast4. Stabilized Mast4 induces increased b-catenin and Runx2 activities, resulting in enhanced osteogenesis of MSCs. DETAILED DESCRIPTION

[0055] In this application, "a" or "an" is used to refer to both singular and plural objects.

[0056] As used herein, administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrently) or sequential administration in any order.

[0057] As used herein, with respect to nucleic acids, proteins, protein fragments or derivatives thereof, the term "biological activity" is defined as the ability of a nucleic acid or amino acid sequence to mimic a known biological function elicited by the wild-type form of the nucleic acid or protein.

[0058] As used herein, the term "bone growth" relates to bone mass. This is due to an increase in the number and size of osteoblasts, as well as an increase in the deposition of osteoid endosteal bone surfaces following systemic administration.

[0059] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Typically, a pharmaceutically acceptable carrier is a pH buffered solution. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Polyethylene glycol (PEG) and

[0060] As used herein, the term "connective tissue" is any tissue that connects and supports other tissues or organs, including but not limited to ligaments, cartilage, tendons, bones, or synovium of a mammalian host.

[0061] The terms "connective tissue cells" or "cells of connective tissue" as used herein include cells present in connective tissue, such as fibroblasts, cartilage cells (chondrocytes) and bone cells (osteoblasts / osteocytes), as well as fat cells (adipocytes) and smooth muscle cells. Preferably, connective tissue cells are fibroblasts, chondrocytes and osteocytes. More preferably, connective tissue cells are fibroblasts. Alternatively, connective tissue cells are osteoblasts or osteocytes. It will be appreciated that the present invention can be practiced using mixed cultures of connective tissue cells as well as cells of a single type. It is also appreciated that tissue cells can be treated (e.g., chemically or irradiated) so that the cells stably express a gene of interest. Preferably, connective tissue cells do not cause a negative immune response when injected into a host organism. It is understood that allogeneic cells can be used in this regard, and autologous cells can be used for cell-mediated gene therapy or somatic cell therapy.

[0062] As used herein, a "connective tissue cell line" includes a plurality of connective tissue cells derived from a common parent cell.

[0063] As used herein, "host cell" includes a single cell or cell culture that can be or was a recipient of a vector of the invention. Host cells include progeny of a single host cell, and progeny are not necessarily completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental or deliberate mutation and / or change.

[0064] As used herein, the term "low bone mass" refers to a condition in which the bone mass level is below the age-specific normal value defined in the World Health Organization "Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994). Report of a World Health Organization Study Group. World Health Organization Technical Series 843" (which is incorporated herein by reference to refer to normal and osteoporotic levels of bone mass) standards. In addition, the term "bone mass" refers to the amount of bone per unit area, sometimes also referred to as bone mineral density.

[0065] As used herein, the term "mammalian host" includes members of the animal kingdom, including, but not limited to, humans.

[0066] As used herein, the term "mature bone" refers to mineralized bone, as opposed to non-mineralized bone (eg, skeletal bone).

[0067] As used herein, the term "osteogenetically effective" refers to an amount that affects the formation and development of mature bone.

[0068] As used herein, the term "osteoprogenitor cells" or "bone progenitor cells" are cells that have the potential to become bone cells and are present in the periosteum and bone marrow. Osteoprogenitor cells are derived from connective tissue progenitor cells, which are also present in peripheral tissues (muscles).

[0069] As used herein, the term "patient" includes members of the animal kingdom, including, but not limited to, humans.

[0070] As used herein, a composition is "pharmacologically or physiologically acceptable" if its administration can be tolerated by the recipient animal and is otherwise suitable for administration to that animal. Such drugs are said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient patient.

[0071] As used herein, "pharmaceutically acceptable carriers and / or diluents" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0072] As used herein, a "promoter" can be any DNA sequence that is active and controls transcription in eukaryotic cells. Promoters can be active in both eukaryotic cells and prokaryotic cells. Preferably, the promoter is active in mammalian cells. The promoter can be constitutively expressed or inducible. Preferably, the promoter is inducible. Preferably, the promoter can be induced by external stimuli. More preferably, the promoter can be induced by hormones or metals. Similarly, an "enhancer component" that controls transcription can also be inserted into a DNA vector construct and used with the construct of the present invention to enhance the expression of the target gene.

[0073] As used herein, a "subject" is a vertebrate, preferably a mammal, more preferably a human.

[0074] As used herein, "dose" refers to a specified amount of a therapeutic agent to be taken at one time or at specified intervals.

[0075] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, alleviation of the extent of the disease, a stable (i.e., non-deteriorating) state of the disease, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and alleviation (whether partial or total), whether detectable or undetectable. "Treatment" can also mean extending survival compared to the expected survival without treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. People in need of treatment include those who already have the disease and those who need to prevent the disease. "Palliating" disease refers to a slowing or prolonging of the time course of the degree of the disease state and / or the alleviation of adverse clinical manifestations and / or the progression compared to the untreated situation.

[0076] As used herein, "vector", "polynucleotide vector", "construct" and "polynucleotide construct" are used interchangeably herein. The polynucleotide vectors of the present invention can be in any of several forms, including but not limited to RNA, DNA, RNA encapsulated in a retroviral coating, DNA encapsulated in an adenoviral coating, DNA packaged in another virus or virus-like form (e.g., herpes simplex and adeno-associated virus (AAV)), DNA encapsulated in liposomes, complexed with polylysine, complexed with synthetic polycationic molecules, complexed with compounds (e.g., polyethylene glycol (PEG)) to immunologically "mask" the molecule and / or extend the half-life, or DNA coupled to non-viral proteins. Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only the bases A, T, C and G, but also any of their analogs or modified forms of these bases (e.g., methylated nucleotides), internucleotide modifications (e.g., uncharged bonds and thioates), the use of sugar analogs, and modified and / or alternative backbone structures (e.g., polyamides).

[0077] The term "antibody" refers to a specific immunoglobulin for an antigenic site. A gene of interest (e.g., encoding GSK-3α or GSK-3βa) is cloned into an expression vector to obtain a protein encoded by the gene, and antibodies can be prepared from the protein according to methods commonly used in the art. The types of antibodies include polyclonal antibodies or monoclonal antibodies, and include all immunoglobulin antibodies. The antibody not only includes a complete form with two full-length light chains and two full-length heavy chains, but also includes a functional fragment of an antibody molecule with a specific antigen binding site (binding domain) for an antigenic site to retain antigen binding function, although they do not have a complete antibody structure containing two light chains and two heavy chains.

[0078] Unless otherwise indicated, the term "polynucleotide" may have the same meaning as nucleotide or nucleic acid, and refers to deoxyribonucleotides or ribonucleotides. Unless otherwise indicated, polynucleotides may include analogs of natural nucleotides and analogs of sugar or base moieties with modifications. Polynucleotides may be modified as needed by the various methods known in the art. The example of modification may include methylation, capping, replacement of natural nucleotides with one or more homologues, and modification between nucleotides, for example, modification to uncharged linkages (for example, methylphosphonates, phosphotriesters, aminophosphorates, carbamates, etc.) or charged linkages (for example, phosphorothioates, phosphorodithioates, etc.).

[0079] In a specific embodiment, as a compound capable of specifically binding to a nucleic acid encoding a protein of interest or a fragment thereof, the polynucleotide capable of specifically binding to a nucleic acid encoding a protein of interest or a fragment thereof can be a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi interacting RNA (piRNA), a small nuclear RNA (snRNA) or an antisense oligonucleotide, each of which is specific to the nucleic acid encoding a protein of interest or a fragment thereof, or a combination thereof.

[0080] In another specific embodiment, the compound capable of specifically binding to a nucleic acid encoding a protein of interest or a fragment thereof may include a polynucleotide capable of specifically binding to a nucleic acid encoding a protein of interest or a fragment thereof, and may be CRISPR-Cas, which includes a guide RNA specific to the nucleic acid encoding the protein of interest or a fragment thereof. In a specific embodiment, Cas may be Cas9.

[0081] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) refers to the sites of many short direct repeat sequences found in the genomes of bacteria or archaea, revealing the gene sequences therein. The CRISPR-Cas system includes Cas9 as an essential protein component, which forms a complex with guide RNA (particularly two RNAs, called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), contained in the guide RNA), and it acts as an active nuclease.

[0082] In a specific embodiment, in order to make the CRISPR-Cas system act specifically on the target gene of interest, the guide RNA can be in the form of a double RNA or a single-stranded guide RNA, wherein the double RNA includes a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) specific to the nucleic acid encoding the protein of interest, and the single-stranded guide RNA includes a portion of the crRNA and the tracrRNA and hybridizes with the nucleic acid encoding the protein of interest. The double RNA and the single-stranded guide RNA can hybridize at least partially with the polynucleotide encoding the protein of interest.

[0083] Specifically, the guide RNA can be a double RNA or a single-stranded guide RNA, the double RNA includes a crRNA and a tracrRNA hybridized with a target sequence selected from a nucleotide sequence encoding a protein of interest, and the single-stranded guide RNA includes a portion of crRNA and tracrRNA and hybridizes with the nucleotides encoding the protein of interest. The gene of interest as the target sequence includes a polynucleotide sequence that is at least partially complementary to crRNA or sgRNA, and a sequence including a protospacer-adjacent motif (PAM). PAM can be a sequence well known in the art, which can have a sequence suitable for recognition by a nuclease protein. The gene of interest targeted by the CRISPR-Cas system can be an endogenous DNA or an artificial DNA. The nucleotides encoding the protein of interest can be specific endogenous DNA of eukaryotic cells, more specifically, endogenous DNA of chondrocytes.

[0084] In a specific embodiment, crRNA or sgRNA may include twenty consecutive polynucleotides complementary to the target DNA. The nucleic acid encoding the Cas9 protein or the Cas9 protein may be a microorganism derived from the genus Streptococcus. The microorganism of the genus Streptococcus may be Streptococcus pyogenes. PAM may represent a 5'-NGG-3' trinucleotide, and the Cas9 protein may also include a nuclear localization signal (NLS) at the C-terminus or N-terminus to improve efficiency.

[0085] In the composition disclosed herein for promoting the production of bones from eukaryotic cells, the eukaryotic cells may be yeast cells, fungal cells, protozoan cells, plant cells, higher plant cells, insect cells, amphibian cells or mammalian cells. The mammals may be different, such as humans, monkeys, cows, horses, pigs, etc. The eukaryotic cells may include cultured cells (in vitro), transplanted cells, in vivo cells or recombinant cells isolated from an individual, but are not limited thereto. The eukaryotic cells isolated from an individual may be eukaryotic cells isolated from the same individual into which the product (including bones) prepared from the eukaryotic cells is injected. In this case, it is advantageous because side effects, such as unnecessary hyperimmune reactions or rejection reactions, including graft-versus-host reactions produced by the injection of products prepared from different individuals, can be prevented.

[0086] In specific embodiments, the eukaryotic cell may be a fibroblast or a chondrocyte or a mesenchymal stem cell or an osteoprogenitor cell (MC3T3-E1; pre-osteoblast).

[0087] MAST4

[0088] MAST4 is a protein derived from humans (Homo sapiens) or mice (Mus musculus), but the same protein can also be expressed in other mammals (eg, monkeys, cows, horses, etc.).

[0089] Human MAST4 may include any one of the twelve isoforms present in human cells. The twelve isoforms may include the following amino acid sequences. The isoform sequences are based on the NCBI reference sequence.

[0090] Isoform 1 - NP_055998.1 1MDESSILRRRR GLQKELSLPR RGSLIDSQKW NCLVKRCRTS NRKSLIGNGQ SPALPRPHSP 61LSAHAGNSPQ DSPRNFSPSA SAHFSFARRT DGRRWSLASL PSSGYGTNTP SSTVSSSCSS 121QEKLHQLPYQ PTPDELHFLS KHFCTTESIA TENRCRNTPM RPRSRSLSPG RSPACCDHEI 181IMMNHVYKER FPKATAQMEE RLKEIITSYS PDNVLPLADG VLSFTHHQII ELARDCLDKS 241HQGLITSRYF LELQHKLDKL LQEAHDRSES GELAFIKQLV RKILIVIARP ARLLECLEFD 301PEEFYYLLEA AEGHAKEGQG IKTDIPRYII SQLGLNKDPL EEMAHLGNYD SGTAETPETD 361ESVSSSNASL KLRRKPREST FETIKLISNG AYGAVYFVRH KESRQRFAMK KINKQNLILR 421NQIQQAFVER DILTFAENPF VVSMYCSFET RRHLCMVMEY VEGGDCATLM KNMGPLPVDM 481ARMYFAETVL ALEYLHNYGI VHRDLKPDNL LVTSMGHIKL TDFGLSKVGL MSMTTNLYEG 541HIEKDAREFL DKQVCGTPEY IAPEVILRQG YGKPVDWWAM GIILYEFLVG CVPFFGDTPE 601ELFGQVISDE INWPEKDEAP PPDAQDLITL LLRQNPLERL GTGGAYEVKQ HRFFRSLDWN 661SLLRQKAEFIPQLESEDDTS YFDTRSEKYH HMETEEEDDT NDEDFNVEIR QFSSCSHRFS 721KVFSSIDRIT QNSAEEKEDS VDKTKSTTLP STETLSWSSE YSEMQQLSTS NSSDTESNRH 781KLSSGLLPKL AISTEGEQDE AASCPGDPHE EPGKPALPPE ECAQEEPEVT TPASTISSST 841LSVGSFSEHL DQINGRSECV DSTDNSSKPS SEPASHMARQ RLESTEKKKI SGKVTKSLSA 901SALSLMIPGD MFAVSPLGSP MSPHSLSSDP SSSRDSSPSR DSSAASASPH QPIVIHSSGK 961NYGFTIRAIR VYVGDSDIYT VHHIVWNVEE GSPACQAGLK AGDLITHING EPVHGLVHTE 1021VIELLLKSGN KVSITTTPFE NTSIKTGPAR RNSYKSRMVR RSKKSKKKES LERRRSLFKK 1081LAKQPSPLLH TSRSFSCLNR SLSSGESLPG SPTHSLSPRS PTPSYRSTPD FPSGTNSSQS 1141SSPSSSAPNS PAGSGHIRPS TLHGLAPKLG GQRYRSGRRK SAGNIPLSPL ARTPSPTPQP 1201TSPQRSPSPL LGHSLGNSKI AQAFPSKMHS PPTIVRHIVR PKSAEPPRSP LLKRVQSEEK 1261LSPSYGSDKK HLCSRKHSLE VTQEEVQREQ SQREAPLQSL DENVCDVPPL SRARPVEQGC 1321LKRPVSRKVG RQESVDDLDR DKLKAKVVVK KADGFPEKQE SHQKSHGPGS DLENFALFKL 1381EEREKKVYPK AVERSSTFEN KASMQEAPPL GSLLKDALHK QASVRASEGA MSDGRVPAEH 1441RQGGGDFRRA PAPGTLQDGL CHSLDRGISG KGEGTEKSSQ AKELLRCEKL DSKLANIDYL 1501RKKMSLEDKE DNLCPVLKPK MTAGSHECLP GNPVRPTGGQ QEPPPASESR AFVSSTHAAQ 1561 MSAVSFVPLK ALTGRVDSGT EKPGLVAPES PVRKSPSEYK LEGRSVSCLKPIEGTLDIAL 1621 LSGPQASKTE LPSPESAQSP SPSGDVRASV PPVLPSSSGK KNDTTSARELSPSSLKMNKS 1681 YLLEPWFLPP SRGLQNSPAV SLPDPEFKRD RKGPHPTARS PGTVMESNPQQREGSSPKHQ 1741 DHTTDPKLLT CLGQNLHSPD LARPRCPLPP EASPSREKPG LRESSERGPPTARSERSAAR 1801 ADTCREPSME LCFPETAKTS DNSKNLLSVG RTHPDFYTQT QAMEKAWAPGGKTNHKDGPG 1861 EARPPPRDNS SLHSAGIPCE KELGKVRRGV EPKPEALLAR RSLQPPGIESEKSEKLSSFP 1921 SLQKDGAKEP ERKEQPLQRH PSSIPPPPLT AKDLSSPAAR QHCSSPSHASGREPGAKPST 1981 AEPSSSPQDP PKPVAAHSES SSHKPRPGPD PGPPKTKHPD RSLSSQKPSVGATKGKEPAT 2041 QSLGGSSREG KGHSKSGPDV FPATPGSQNK ASDGIGQGEG GPSVPLHTDRAPLDAKPQPT 2101 SGGRPLEVLE KPVHLPRPGH PGPSEPADQK LSAVGEKQTL SPKHPKPSTVKDCPTLCKQT 2161 DNRQTDKSPS QPAANTDRRA EGKKCTEALY APAEGDKLEA GLSFVHSENRLKGAERPAAG 2221 VGKGFPEARG KGPGPQKPPT EADKPNGMKR SPSATGQSSF RSTALPEKSLSCSSSFPETR 2281 AGVREASAAS SDTSSAKAAG GMLELPAPSN RDHRKAQPAG EGRTHMTKSDSLPSFRVSTL 2341 PLESHHPDPN TMGGASHRDR ALSVTATVGE TKGKDPAPAQ PPPARKQNVGRDVTKPSPAP 2401 NTDRPISLSN EKDFVVRQRR GKESLRSSPH KKAL(SEQ ID NO:1),

[0091] Isotype 2 - NP_942123.1 1 MGEKVSEAPE PVPRGCSGHG SRTPASALVA ASSPGASSAE SSSGSETLSE EGEPGGFSRE 61 HQPPPPPPLG GTLGARAPAA WAPASVLLER GVLALPPPLP GGAVPPAPRG SSASQEEQDE 121 ELDHILSPPP MPFRKCSNPD VASGPGKSLK YKRQLSEDGR QLRRGSLGGA LTGRYLLPNP 181 VAGQAWPASA ETSNLVRMRS QALGQSAPSL TASLKELSLP RRGSLIDSQK WNCLVKRPVC 241 PNAGRTSPLG(SEQ ID NO:2),

[0092] Isotype 3 - NP_001158136.1 1 MGEKVSEAPE PVPRGCSGHG SRTPASALVA ASSPGASSAE SSSGSETLSE EGEPGGFSRE 61 HQPPPPPPLG GTLGARAPAA WAPASVLLER GVLALPPPLP GGAVPPAPRG SSASQEEQDE 121 ELDHILSPPP MPFRKCSNPD VASGPGKSLK YKRQLSEDGR QLRRGSLGGA LTGRYLLPNP 181 VAGQAWPASA ETSNLVRMRS QALGQSAPSL TASLKELSLP RRGSFCRTSN RKSLIGNGQS 241 PALPRPHSPL SAHAGNSPQD SPRNFSPSAS AHFSFARRTD GRRWSLASLP SSGYGTNTPS 301 STVSSSCSSQ EKLHQLPYQP TPDELHFLSK HFCTTESIAT ENRCRNTPMR PRSRSLSPGR 361 SPACCDHEII MMNHVYKERF PKATAQMEER LKEIITSYSP DNVLPLADGV LSFTHHQIIE 421 LARDCLDKSH QGLITSRYFL ELQHKLDKLL QEAHDRSESG ELAFIKQLVR KILIVIARPA 481 RLLECLEFDP EEFYYLLEAA EGHAKEGQGI KTDIPRYIIS QLGLNKDPLE EMAHLGNYDS 541 GTAETPETDE SVSSSNASLK LRRKPRESDF ETIKLISNGA YGAVYFVRHK ESRQRFAMKK 601 INKQNLILRN QIQQAFVERD ILTFAENPFV VSMYCSFETR RHLCMVMEYV EGGDCATLMK 661 NMGPLPVDMA RMYFAETVLA LEYLHNYGIV HRDLKPDNLL VTSMGHIKLT DFGLSKVGLM 721 SMTTNLYEGH IEKDAREFLD KQVCGTPEYI APEVILRQGY GKPVDWWAMG IILYEFLVGC 781 VPFFGDTPEE LFGQVISDEI NWPEKDEAPP PDAQDLITLL LRQNPLERLG TGGAYEVKQH 841 RFFRSLDWNS LLRQKAEFIP QLESEDDTSY FDTRSEKYHH METEEEDDTN DEDFNVEIRQ 901 FSSCSHRFSK VFSSIDRITQ NSAEEKEDSV DKTKSTTLPS TETLSWSSEY SEMQQLSTSN 961 SSDTESNRHK LSSGLLPKLA ISTEGEQDEA ASCPGDPHEE PGKPALPPEE CAQEEPEVTT 1021 PASTISSSTL SVGSFSEHLD QINGRSECVD STDNSSKPSS EPASHMARQR LESTEKKKIS 1081 GKVTKSLSAS ALSLMIPGDM FAVSPLGSPM SPHSLSSDPS SSRDSSPSRDSSAASASPHQ 1141 PIVIHSSGKN YGFTIRAIRV YVGDSDIYTV HHIVWNVEEG SPACQAGLKAGDLITHINGE 1201 PVHGLVHTEV IELLLKSGNK VS ITTTPFEN TS IKTGPARR NSYKSRMVRRSKKSKKKESL 1261 ERRRSLFKKL AKQPSPLLHT SRSFSCLNRS LSSGESLPGS PTHSLSPRSPTPSYRSTPDF 1321 PSGTNSSQSS SPSSSAPNSP AGSGHIRPST LHGLAPKLGG QRYRSGRRKS AGNIPLSPLA 1381 RTPSPTPQPT SPQRSPSPLL GHSLGNSKIA QAFPSKMHSP PTIVRHIVRPKSAEPPRSPL 1441 LKRVQSEEKL SPSYGSDKKH LCSRKHSLEV TQEEVQREQS QREAPLQSLDENVCDVPPLS 1501 RARPVEQGCL KRPVSRKVGR QESVDDLDRD KLKAKVVVKK ADGFPEKQE SHQKSHGPGSD 1561 LENFALFKLE EREKKVYPKA VERSSTFENK ASMQEAPPLG SLLKDALHKQASVRASEGAM 1621 SDGRVPAEHR QGGGDFRRAP APGTLQDGLC HSLDRGI SGK GEGTEKSSQAKELLRCEKLD 1681 SKLANI DYLR KKMSLEDKED NLCPVLKPKM TAGSHECLPG NPVRPTGGQQEPPPASESRA 1741 FVSSTHAAQM SAVSFVPLKA LTGRVDSGTE KPGLVAPESP VRKSPSEYKLEGRSVSCLKP 1801 IEGTLDIALL SGPQASKTEL PSPESAQSPS PSGDVRASVP PVLPSSSGKKNDTTSARELS 1861 PSSLKMNKSY LLEPWFLPPS RGLQNSPAVS LPDPEFKRDR KGPHPTARSPGTVMESNPQQ 1921 REGSSPKHQD HTTDPKLLTC LGQNLHSPDL ARPRCPLPPE ASPSREKPGLRESSERGPPT 1981 ARSERSAARA DTCREPSMEL CFPETAKTSD NSKNLLSVGR THPDFYTQTQAMEKAWAPGG 2041 KTNHKDGPGE ARPPPRDNSS LHSAGIPCEK ELGKVRRGVE PKPEALLARRSLQPPGIESE 2101 KSEKLSSFPS LQKDGAKEPE RKEQPLQRHP SSIPPPPLTA KDLSSPAARQHCSSPSHASG 2161 REPGAKPSTA EPSSSPQDPP KPVAAHSESS SHKPRPGPDP GPPKTKHPDRSLSSQKPSVG 2221 ATKGKEPATQ SLGGSSREGK GHSKSGPDVF PATPGSQNKA SDGIGQGEGGPSVPLHTDRA 2281 PLDAKPQPTS GGRPLEVLEK PVHLPRPGHP GPSEPADQKL SAVGEKQTLSPKHPKPSTVK 2341 DCPTLCKQTD NRQTDKSPSQ PAANTDRRAE GKKCTEALYA PAEGDKLEAGLSFVHSENRL 2401KGAERPAAGV GKGFPEARGK GPGPQKPPTE ADKPNGMKRS PSATGQSSFR STALPEKSLS 2461CSSSFPETRA GVREASAASSDTSSAKAAGG MLELPAPSNR DHRKAQPAGE GRTHMTKSDS 2521LPSFRVSTLP LESHHPDPNT MGGASHRDRA LSVTATVGET KGKDPAPAQP PPARKQNVGR 2581DVTKPSPAPN TDRPISLSNE KDFVVRQRRG KESLRSSPHK KAL(SEQ ID NO:3),

[0093] Isotype 4 - NP_001277155.1 1MKAQRERLQIPGLTLDCRTS NRKSLIGNGQ SPALPRPHSP LSAHAGNSPQ DSPRNFSPSA 61SAHFSFARRN DRTDGRRWSL ASLPSSGYGT NTPSSTVSSS CSSQEKLHQL PYQPTPDELH 121FLSKHFCTTE SIATENRCRN TPMRPRSRSL SPGRSPACCD HEIIMMNHVY KERFPKATAQ 181MEERLKEIIT SYSPDNVLPL ADGVLSFTHH QIIELARDCL DKSHQGLITS RYFLELQHKL 241DKLLQEAHDR SESGELAFIK QLVRKILIVI ARPARLLECL EFDPEEFYYL LEAAEGHAKE 301GQGIKTDIPR YIISQLGLNK DPLEEMAHLG NYDSGTAETP ETDESVSSSN ASLKLRRKPR 361ESDFETIKLI SNGAYGAVYF VRHKESRQRF AMKKINKQNL ILRNQIQQAF VERDILTFAE 421NPFVVSMYCS FETRRHLCMV MEYVEGGDCA TLMKNMGPLP VDMARMYFAE TVLALEYLHN 481YGIVHRDLKP DNLLVTSMGH IKLTDFGLSK VGLMSMTTNL YEGHIEKDAR EFLDKQVCGT 541PEYIAPEVIL RQGYGKPVDW WAMGIILYEF LVGCVPFFGD TPEELFGQVI SDEINWPEKD 601EAPPPDAQDL ITLLLRQNPL ERLGTGGAYE VKQHRFFRSL DWNSLLRQKA EFIPQLESED 661DTSYFDTRSE KYHHMETEEE DDTNDEDFNV EIRQFSSCSH RFSKVFSSID RITQNSAEEK 721EDSVDKTKST TLPSTETLSW SSEYSEMQQL STSNSSDTES NRHKLSSGLL PKLAISTEGE 781QDEAASCPGD PHEEPGKPAL PPEECAQEEP EVTTPASTIS SSTLSVGSFS EHLDQINGRS 841ECVDSTDNSS KPSSEPASHM ARQRLESTEK KKISGKVTKS LSASALSLMI PGDMFAVSPL 901GSPMSPHSLSSDPSSSRDSS PSRDSSAASA SPHQPIVIHS SGKNYGFTIR AIRVYVGDSD 961IYTVHHIVWN VEEGSPACQA GLKAGDLITH INGEPVHGLV HTEVIELLLK SGNKVSITTT 1021PFENTSIKTG PARRNSYKSR MVRRSKKSKK KESLERRRSL FKKLAKQPSP LLHTSRSFSC 1081LNRSLSSGES LPGSPTHSLS PRSPTPSYRS TPDFPSGTNS SQSSSPSSSA PNSPAGSGHI 1141RPSTLHGLAP KLGGQRYRSG RRKSAGNIPL SPLARTPSPT PQPTSPQRSP SPLLGHSLGN 1201SKIAQAFPSK MHSPPTIVRH IVRPKSAEPP RSPLLKRVQS EEKLSPSYGS DKKHLCSRKH 1261SLEVTQEEVQ REQSQREAPL QSLDENVCDV PPLSRARPVE QGCLKRPVSR KVGRQESVDD 1321LDRDKLKAKV VVKKADGFPE KQESHQKSHG PGSDLENFAL FKLEEREKKV YPKAVERSST 1381FENKASMQEA PPLGSLLKDA LHKQASVRAS EGAMSDGRVP AEHRQGGGDF RRAPAPGTLQ 1441DGLCHSLDRG ISGKGEGTEK SSQAKELLRC EKLDSKLANI DYLRKKMSLE DKEDNLCPVL 1501KPKMTAGSHE CLPGNPVRPT GGQQEPPPAS ESRAFVSSTH AAQMSAVSFV PLKALTGRVD 1561SGTEKPGLVA PESPVRKSPS EYKLEGRSVS CLKPIEGTLD IALLSGPQAS KTELPSPESA 1621QSPSPSGDVR ASVPPVLPSS SGKKNDTTSA RELSPSSLKM NKSYLLEPWF LPPSRGLQNS 1681PAVSLPDPEF KRDRKGPHPT ARSPGTVMES NPQQREGSSP KHQDHTTDPK LLTCLGQNLH 1741SPDLARPRCP LPPEASPSRE KPGLRESSER GPPTARSERS AARADTCREP SMELCFPETA 1801KTSDNSKNLL SVGRTHPDFY TQTQAMEKAW APGGKTNHKD GPGEARPPPR DNSSLHSAGI 1861PCEKELGKVR RGVEPKPEAL LARRSLQPPG IESEKSEKLS SFPSLQKDGA KEPERKEQPL 1921QRHPSSIPPP PLTAKDLSSP AARQHCSSPS HASGREPGAK PSTAEPSSSP QDPPKPVAAH 1981SESSSHKPRP GPDPGPPKTK HPDRSLSSQK PSVGATKGKE PATQSLGGSS REGKGHSKSG 2041PDVFPATPGS QNKASDGIGQ GEGGPSVPLH TDRAPLDAKP QPTSGGRPLE VLEKPVHLPR 2101PGHPGPSEPA DQKLSAVGEK QTLSPKHPKP STVKDCPTLC KQTDNRQTDK SPSQPAANTD 2161RRAEGKKCTE ALYAPAEGDK LEAGLSFVHS ENRLKGAERP AAGVGKGFPE ARGKGPGPQK 2221PPTEADKPNG MKRSPSATGQ SSFRSTALPE KSLSCSSSFP ETRAGVREAS AASSDTSSAK 2281AAGGMLELPA PSNRDHRKAQ PAGEGRTHMT KSDSLPSFRV STLPLESHHP DPNTMGGASH 2341RDRALSVTAT VGETKGKDPA PAQPPPARKQ NVGRDVTKPS PAPNTDRPIS LSNEKDFVVR 2401QRRGKESLRS SPHKKAL(SEQ ID NO:4),

[0094] Isotype 5 - NP_001277156.1 1MDMSDPNFWT VLSNFTLPHL RSGNRLRRTQ SCRTSNRKSL IGNGQSPALP RPHSPLSAHA 61GNSPQDSPRN FSPSASAHFS FARRTDGRRW SLASLPSSGY GTNTPSSTVS SSCSSQEKLH 121QLPYQPTPDE LHFLSKHFCT TESIATENRC RNTPMRPRSR SLSPGRSPAC CDHEIIMMNH 181VYKERFPKAT AQMEERLKEI ITSYSPDNVL PLADGVLSFT HHQIIELARD CLDKSHQGLI 241TSRYFLELQH KLDKLLQEAH DRSESGELAF IKQLVRKILI VIARPARLLE CLEFDPEEFY 301YLLEAAEGHA KEGQGIKTDIPRYIISQLGL NKDPLEEMAH LGNYDSGTAE TPETDESVSS 361SNASLKLRRK PRESDFETIK LISNGAYGAV YFVRHKESRQ RFAMKKINKQ NLILRNQIQQ 421AFVERDILTF AENPFVVSMY CSFETRRHLC MVMEYVEGGD CATLMKNMGP LPVDMARMYF 481AETVLALEYL HNYGIVHRDL KPDNLLVTSM GHIKLTDFGL SKVGLMSMTT NLYEGHIEKD 541AREFLDKQVC GTPEYIAPEV ILRQGYGKPV DWWAMGIILY EFLVGCVPFF GDTPEELFGQ 601VISDEINWPE KDEAPPPDAQ DLITLLLRQN PLERLGTGGA YEVKQHRFFR SLDWNSLLRQ 661KAEFIPQLES EDDTSYFDTR SEKYHHMETE EEDDTNDEDF NVEIRQFSSC SHRFSKVFSS 721IDRITQNSAE EKEDSVDKTK STTLPSTETL SWSSEYSEMQ QLSTSNSSDT ESNRHKLSSG 781LLPKLAISTE GEQDEAASCP GDPHEEPGKP ALPPEECAQE EPEVTTPAST ISSSTLSDMF 841AVSPLGSPMS PHSLSSDPSS SRDSSPSRDS SAASASPHQP IVIHSSGKNY GFTIRAIRVY 901VGDSDIYTVH HIVWNVEEGS PACQAGLKAG DLITHINGEP VHGLVHTEVI ELLLKSGNKV 961SITTTPFENT SIKTGPARRN SYKSRMVRRS KKSKKKESLE RRRSLFKKLA KQPSPLLHTS 1021RSFSCLNRSL SSGESLPGSP THSLSPRSPT PSYRSTPDFP SGTNSSQSSS PSSSAPNSPA 1081GSGHIRPSTL HGLAPKLGGQ RYRSGRRKSA GNIPLSPLAR TPSPTPQPTS PQRSSPPLLG 1141HSLGNSKIAQ AFPSKMHSPP TIVRHIVRPK SAEPPRSPLL KRVQSEEKLS PSYGSDKKHL 1201CSRKHSLEVT QEEVQREQSQ REAPLQSLDE NVCDVPPLSR ARPVEQGCLK RPVSRKVGRQ 1261ESVDDLDRDK LKAKVVVKKA DGFPEKQESH QKSHGPGSDL ENFALFKLEE REKKVYPKAV 1321ERSSTFENKA SMQEAPPLGS LLKDALHKQA SVRASEGAMSDGRWPAEHRQ GGGDFRRAPA 1381PGTLQDGLCH SLDRGISGKG EGTEKSSQAK ELLRCEKLDS KLANIDYLRK KMSLEDKEDN 1441LCPVLKPKMT AGSHECLPGN PVRPTGGQQE PPPASESRAF VSSTHAAQMS AVSFVPLKAL 1501TGRVDSGTEK PGLVAPESPV RKSPSEYKLE GRSVSCLKPI EGTLDIALLS GPQASKTELP 1561SPESAQSPSP SGDVRASVPP VLPSSSGKKN DTTSARELSP SSLKMNKSYL LEPWFLPPSR 1621GLQNSPAVSL PDPEFKRDRK GPHPTARSPG TVMESNPQQR EGSSPKHQDH TTDPKLLTCL 1681GQNLHSPDLA RPRCPLPPEA SPSREKPGLR ESSERGPPTA RSERSAARAD TCREPSMELC 1741FPETAKTSDN SKNLLSVGRT HPDFYTQTQA MEKAWAPGGK TNHKDGPGEA RPPPRDNSSL 1801HSAGIPCEKE LGKVRRGVEP KPEALLARRS LQPPGIESEK SEKLSSFPSL QKDGAKEPER 1861KEQPLQRHPS SIPPPPLTAK DLSSPAARQH CSSPSHASGR EPGAKPSTAE PSSSPQDPPK 1921PVAAHSESSS HKPRPGPDPG PPKTKHPDRS LSSQKPSVGA TKGKEPATQS LGGSSREGKG 1981HSKSGPDVFP ATPGSQNKASDGIGQGEGGPSVPLHTDRAP LDAKPQPTSG GRPLEVLEKP 2041VHLPRPGHPGPSEPADQKLS AVGEKQTLSP KHPKPSTVKD CPTLCKQTDN RQTDKSPSQP 2101AANTDRRAEG KKCTEALYAP AEGDKLEAGL SFVHSENRLK GAERPAAGVG KGFPEARGKG 2161PGPQKPPTEA DKPNGMKRSP SATGQSSFRS TALPEKSLSC SSSFPETRAG VREASAASSD 2221TSSAKAAGGM LELPAPSNRD HRKAQPAGEG RTHMTKSDSL PSFRVSTLPL ESHHPDPNTM 2281GGASHRDRAL SVTATVGEK GKDPAPAQPP PARKQNVGRD VTKPSPAPNT DRPISLSNEK 2341DFVVRQRRGK ESLRSSPHKK AL(SEQ ID NO:5),

[0095] Isotype 6 - NP_001277157.1 1MGEKVSEAPE PVPRGCSGHG SRTPASALVA ASSPGASSAE SSSGSETLSE EGEPGGFSRE 61HQPPPPPPLG GTLGARAPAA WAPASVLLER GVLALPPPLP GGAVPPAPRG SSASQEEQDE 121ELDHILSPPP MPFRKCSNPD VASGPGKSLK YKRQLSEDGR QLRRGSLGGA LTGRYLLPNP 181VAGQAWPASA ETSNLVRMRS QALGQSAPSL TASLKELSLP RRGSFPVCPN AGRTSPLG(SEQ ID NO:6),

[0096] Isotype 7 - NP_001284580.1 1 MDMSDPNFWT VLSNFTLPHL RSGNRLRRTQ SCRTSNRKSL IGNGQSPALP RPHSPLSAHA 61 GNSPQDSPRN FSPSASAHFS FARRTDGRRW SLASLPSSGY GTNTPSSTVS SSCSSQEKLH 121 QLPYQPTPDE LHFLSKHFCT TESIATENRC RNTPMRPRSR SLSPGRSPAC CDHE IIMMNH 181 VYKERFPKAT AQMEERLKE I ITSYSPDNVL PLADGVLSFT HHQIIELARDCLDKSHQGLI 241 TSRYFLELQH KLDKLLQEAH DRSESGELAF IKQLVRKILI VIARPARLLE CLEFDPEEFY 301 YLLEAAEGHA KEGQGIKTDI PRYIISQLGL NKDPLEEMAH LGNYDSGTAE TPETDESVSS 361 SNASLKLRRK PRESDFETIK LISNGAYGAV YFVRHKESRQ RFAMKKINKQ NLILRNQIQQ 421 AFVERDILTF AENPFVVSMY CSFETRRHLC MVMEYVEGGD CATLMKNMGP LPVDMARMYF 481 AETVLALEYL HNYGIVHRDL KPDNLLVTSM GHIKLTDFGL SKVGLMSMTT NLYEGHIEKD 541 AREFLDKQVC GTPEYIAPEV ILRQGYGKPV DWWAMGIILY EFLVGCVPFF GDTPEELFGQ 601 VISDEINWPE KDEAPPPDAQ DLITLLLRQN PLERLGTGGA YEVKQHRFFR SLDWNSLLRQ 661 KAEFIPQLES EDDTSYFDTR SEKYHHMETE EEDDTNDEDF NVEIRQFSSC SHRFSKVFSS 721 IDRITQNSAE EKEDSVDKTK STTLPSTETL SWSSEYSEMQ QLSTSNSSDT ESNRHKLSSG 781 LLPKLAISTE GEQDEAASCP GDPHEEPGKP ALPPEECAQE EPEVTTPAST ISSSTLSVGS 841 FSEHLDQING RSECVDSTDN SSKPSSEPAS HMARQRLEST EKKKISGKVT KSLSASALSL 901 MIPGDMFAVS PLGSPMSPHS LSSDPSSSRD SSPSRDSSAA SASPHQPIVI HSSGKNYGFT 961 IRAIRVYVGD SDIYTVHHIV WNVEEGSPAC QAGLKAGDLI THINGEPVHG LVHTEVIELL 1021 LKSGNKVSIT TTPFENTSIK TGPARRNSYK SRMVRRSKKS KKKESLERRRSLFKKLAKQP 1081 SPLLHTSRSF SCLNRSLSSG ESLPGSPTHS LSPRSPTPSY RSTPDFPSGTNSSQSSSPSS 1141 SAPNSPAGSG HIRPSTLHGL APKLGGQRYR SGRRKSAGNI PLSPLARTPSPTPQPTSPQR 1201 SPSPLLGHSL GNSKIAQAFP SKMHSPPTIV RHIVRPKSAE PPRSPLLKRVQSEEKLSPSY 1261 GSDKKHLCSR KHSLEVTQEE VQREQSQREA PLQSLDENVC DVPPLSRARPVEQGCLKRPV 1321 SRKVGRQESV DDLDRDKLKA KVVVKKADGF PEKQESHQKS HGPGSDLENFALFKLEEREK 1381 KVYPKAVERS STFENKASMQ EAPPLGSLLK DALHKQASVR ASEGAMSDGRVPAEHRQGGG 1441 DFRRAPAPGT LQDGLCHSLD RGISGKGEGT EKSSQAKELL RCEKLDSKLA NIDYLRKKMS 1501 LEDKEDNLCP VLKPKMTAGS HECLPGNPVR PTGGQQEPPP ASESRAFVSSTHAAQMSAVS 1561 FVPLKALTGR VDSGTEKPGL VAPESPVRKS PSEYKLEGRS VSCLKPIEGTLDIALLSGPQ 1621 ASKTELPSPE SAQSPSPSGD VRASVPPVLP SSSGKKNDTT SARELSPSSLKMNKSYLLEP 1681 WFLPPSRGLQ NSPAVSLPDP EFKRDRKGPH PTARSPGTVM ESNPQQREGSSPKHQDHTTD 1741 PKLLTCLGQN LHSPDLARPR CPLPPEASPS REKPGLRESS ERGPPTARSERSAARADTCR 1801 EPSMELCFPE TAKTSDNSKN LLSVGRTHPD FYTQTQAMEK AWAPGGKTNHKDGPGEARPP 1861PRDNSSLHSA GIPCEKELGK VRRGVEPKPE ALLARRSLQP PGIESEKSEK LSSFPSLQKD 1921GAKEPERKEQ PLQRHPSSIP PPPLTAKDLS SPAARQHCSS PSHASGREPG AKPSTAEPSS 1981SPQDPPKPVA AHSESSSHKP RPGPDPGPPK TKHPDRSLSS QKPSVGATKG KEPATQSLGG 2041SSREGKGHSK SGPDVFPATP GSQNKASDGI GQGEGGPSVP LHTDRAPLDA KPQPTSGGRP 2101LEVLEKPVHL PRPGHPGPSE PADQKLSAVG EKQTLSPKHP KPSTVKDCPT LCKQTDNRQT 2161DKSPSQPAAN TDRRAEGKKC TEALYAPAEG DKLEAGLSFV HSENRLKGAE RPAAGVGKGF 2221PEARGKGPGP QKPPTEADKP NGMKRSPSAT GQSSFRSTAL PEKSLSCSSS FPETRAGVRE 2281ASAASSDTSS AKAAGGMLEL PAPSNRDHRK AQPAGEGRTH MTKSDSLPSF RVSTLPLESH 2341HPDPNTMGGA SHRDRALSVT ATVGETKGKD PAPAQPPPAR KQNVGRDVTK PSPAPNTDRP 2401ISLSNEKDFV VRQRRGKESL RSSPHKKAL(SEQ ID NO:7),

[0097] Isotype 8 - NP_001380453.1 1MGEKVSEAPE PVPRGCSGHG SRTPASALVA ASSPGASSAE SSSGSETLSE EGEPGGFSRE 61HQPPPPPPLG GTLGARAPAA WAPASVLLER GVLALPPPLP GGAVPPAPRG SSASQEEQDE 121ELDHILSPPP MPFRKCSNPD VASGPGKSLK YKRQLSEDGR QLRRGSLGGA LTGRYLLPNP 181VAGQAWPASA ETSNLVRMRS QALGQSAPSL TASLKELSLP RRGSFCRTSN RKSLIGNGQS 241PALPRPHSPL SAHAGNSPQD SPRNFSPSAS AHFSFARRND RTDGRRWSLA SLPSSGYGTN 301TPSSTVSSSC SSQEKLHQLP YQPTPDELHF LSKHFCTTES IATENRCRNT PMRPRSRSLS 361PGRSPACCDH EIIMMNHVYK ERFPKATAQM EERLKEIITS YSPDNVLPLA DGVLSFTHHQ 421IIELARDCLD KSHQGLITSR YFLELQHKLD KLLQEAHDRS ESGELAFIKQ LVRKILIVIA 481RPARLLECLE FDPEEFYYLL EAAEGHAKEG QGIKTDIPRY IISQLGLNKD PLEMAHLGN 541YDSGTAETPE TDESVSSSNA SLKLRRKPRE SDFETIKLIS NGAYGAVYFV RHKESRQRFA 601MKKINKQNLI LRNQIQQAFV ERDILTFAEN PFVVSMYCSF ETRRHLCMVM EYVEGGDCAT 661LMKNMGPLPV DMARMYFAET VLALEYLHNY GIVHRDLKPD NLLVTSMGHI KLTDFGLSKV 721GLMSMTTNLY EGHIEKDARE FLDKQVCGTP EYIAPEVILR QGYGKPVDWW AMGIILYEFL 781VGCVPFFGDT PEELFGQVISDEINWPEKDE APPPDAQDLI TLLLRQNPLE RLGTGGAYEV 841KQHRFFRSLD WNSLLRQKAE FIPQLESEDD TSYFDTRSEK YHHMETEEED DTNDEDFNVE 901IRQFSSCSHR FSKVFSSIDR ITQNSAEEKE DSVDKTKSTT LPSTETLSWS SEYSEMQQLS 961TSNSSDTESN RHKLSSGLLP KLAISTEGEQ DEAASCPGDP HEEPGKPALP PEECAQEEPE 1021VTTPASTISS STLSVGSFSE HLDQINGRSE CVDSTDNSSK PSSEPASHMA RQRLESTEKK 1081KISGKVTKSL SASALSLMIP GDMFAVSPLG SPMSPHSLSDPSSSRDSSP SRDSSAASAS 1141PHQPIVIHSS GKNYGFTIRA IRVYVGDSDI YTVHHIVWNV EEGSPACQAG LKAGDLITHI 1201NGEPVHGLVH TEVIELLLKS GNKVSITTTP FENTSIKTGP ARRNSYKSRM VRRSKKSKKK 1261ESLERRRSLF KKLAKQPSPL LHTSRSFSCL NRSLSSGESL PGSPTHSLSP RSPTPSYRST 1321PDFPSGTNSS QSSSPSSSAP NSPAGSGHIR PSTLHGLAPK LGGQRYRSGR RKSAGNIPLS 1381PLARTPSPTP QPTSPQRSPS PLLGHSLGNS KIAQAFPSKM HSPTIVRHI VRPKSAEPPR 1441SPLLKRVQSE EKLSPSYGSD KKHLCSRKHS LEVTQEEVQR EQSQREAPLQ SLDENVCDVP 1501PLSRARPVEQ GCLKRPVSRK VGRQESVDDL DRDKLKAKVV VKKADGFPEK QESHQKSHGP 1561GSDLENFALF CLOTHING PKAVERSSTF ENKASMQEAP PLGSLLKDAL HKQASVRASE 1621GAMSDGRVPA EHRQGGGDFR RAPAPGTLQD GLCHSLDRGI SGKGEGTEKS SQAKELLRCE 1681KLDSKLANID YLRKKMSLED KEDNLCPVLK PKMTAGSHEC LPGNPVRPTG GQQEPPPASE 1741SRAFVSSTHA AQMSAVSFVP LKALTGRVDS GTEKPGLVAP ESPVRKSPSE YKLEGRSVSC 1801LKPIEGTLDI ALLSGPQASK TELPSPESAQ SPSPSGDVRA SVPPVLPSSS GKKNDTTSAR 1861ELSPSSLKMN KSYLLEPWFL PPSRGLQNSP AVSLPDPEFK RDRKGPHPTA RSPGTVMESN 1921PQQREGSSPK HQDHTTDPKL LTCLGQNLHS PDLARPRCPL PPEASPSREK PGLRESSERG 1981PPTARSERSA ARADTCREPS MELCFPETAK TSDNSKNLLS VGRTHPDFYT QTQAMEKAWA 2041PGGKTNHKDG PGEARPPPRD NSSLHSAGIP CEKELGKVRR GVEPKPEALL ARRSLQPPGI 2101ESEKSEKLSS FPSLQKDGAK EPERKEQPLQ RHPSIPPPP LTAKDLSSPA ARQHCSSPSH 2161ASGREPGAKP STAEPSSSPQ DPPKPVAAHS ESSSHKPRPG PDPGPPKTKH PDRSLSSQKP 2221SVGATKGKEP ATQSLGGSSR EGKGHSKSGP DVFPATPGSQ NKASDGIGQG EGGPSVPLHT 2281DRAPLDAKPQ PTSGGRPLEV LEKPVHLPRP GHPGPSEPAD QKLSAVGEKQ TLSPKHPKPS 2341TVKDCPTLCK QTDNRQTDKS PSQPAANTDR RAEGKKCTEA LYAPAEGDKL EAGLSFVHSE 2401NRLKGAERPA AGVGKGFPEA RGKGPGPQKP PTEADKPNGM KRSPSATGQS SFRSTALPEK 2461SLSCSSSFPE TRAGVREASA ASSDTSSAKA AGGMLELPAP SNRDHRKAQP AGEGRTHMTK 2521SDSLPSFRVS TLPLESHHPD PNTMGGASHR DRALSVTATV GETKGKDPAP AQPPPARKQN 2581VGRDVTKPSP APNTDRPISL SNEKDFVVRQ RRGKESLRSS PHKKAL(SEQ ID NO:8),

[0098] Isotype 9 - NP_001380454.1 1MGEKVSEAPE PVPRGCSGHG SRTPASALVA ASSPGASSAE SSSGSETLSE EGEPGGFSRE 61HQPPPPPPLG GTLGARAPAA WAPASVLLER GVLALPPPLP GGAVPPAPRG SSASQEEQDE 121ELDHILSPPP MPFRKCSNPD VASGPGKSLK YKRQLSEDGR QLRRGSLGGA LTGRYLLPNP 181VAGQAWPASA ETSNLVRMRS QALGQSAPSL TASLKELSLP RRGSFCRTSN RKSLIGNGQS 241PALPRPHSPL SAHAGNSPQD SPRNFSPSAS AHFSFARRTD GRRWSLASLP SSGYGTNTPS 301STVSSSCSSQ EKLHQLPYQP TPDELHFLSK HFCTTESIAT ENRCRNTPMR PRSRSLSPGR 361 SPACCDHEII MMNHVYKERF PKATAQMEER LKEIITSYSP DNVLPLADGV LSFTHHQIIE 421 LARDCLDKSH QGLITSRYFL ELQHKLDKLL QEAHDRSESG ELAFIKQLVR KILIVIARPA 481 RLLECLEFDP EEFYYLLEAA EGHAKEGQGI KTDIPRYIIS QLGLNKDPLE EMAHLGNYDS 541 GTAETPETDE SVSSSNASLK LRRKPRESDF ETIKLISNGA YGAVYFVRHK ESRQRFAMKK 601 INKQNLILRN QIQQAFVERD ILTFAENPFV VSMYCSFETR RHLCMVMEYV EGGDCATLMK 661 NMGPLPVDMA RMYFAETVLA LEYLHNYGIV HRDLKPDNLL VTSMGHIKLT DFGLSKVGLM 721 SMTTNLYEGH IEKDAREFLD KQVCGTPEYI APEVILRQGY GKPVDWWAMG IILYEFLVGC 781 VPFFGDTPEE LFGQVISDEI NWPEKDEAPP PDAQDLITLL LRQNPLERLG TGGAYEVKQH 841 RFFRSLDWNS LLRQKAEFIP QLESEDDTSY FDTRSEKYHH METEEEDDTN DEDFNVEIRQ 901 FSSCSHRFSK VFSSIDRITQ NSAEEKEDSV DKTKSTTLPS TETLSWSSEY SEMQQLSTSN 961 SSDTESNRHK LSSGLLPKLA ISTEGEQDEA ASCPGDPHEE PGKPALPPEE CAQEEPEVTT 1021 PASTI SSSTL SDMFAVSPLG SPMSPHSLSS DPSSSRDSSP SRDSSAASASPHQPIVIHSS 1081 GKNYGFTIRA IRVYVGDSDI YTVHHIVWNV EEGSPACQAG LKAGDLITHINGEPVHGLVH 1141 TEVIELLLKS GNKVS ITTTP FENTS IKTGP ARRNSYKSRM VRRSKKSKKKESLERRRSLF 1201 KKKLAKQPSPL LHTSRSFSCL NRSLSSGESL PGSPTHSLSP RSPTPSYRSTPDFPSGTNSS 1261 QSSSPSSSAP NSPAGSGHIR PSTLHGLAPK LGGQRYRSGR RKSAGNI PLSPLARTPSPTP 1321 QPTSPQRSPS PLLGHSLGNS KIAQAFPSKM HSPPTIVRHI VRPKSAEPPRSPLLKRVQSE 1381 EKLSPSYGSD KKHLCSRKHS LEVTQEEVQR EQSQREAPLQ SLDENVCDVPPLSRARPVEQ 1441 GCLKRPVSRK VGRQESVDDL DRDKLKAKVV VKKADGFPEK QESHQKSHGPGSDLENFALF 1501 KLEEREKKVY PKAVERSSTF ENKASMQEAP PLGSLLKDAL HKQASVRASEGAMSDGRVPA 1561 EHRQGGGDFR RAPAPGTLQD GLCHSLDRGI SGKGEGTEKS SQAKELLRCE KLDSKLANID 1621 YLRKKMSLED KEDNLCPVLK PKMTAGSHEC LPGNPVRPTG GQQEPPPASESRAFVSSTHA 1681 AQMSAVSFVP LKALTGRVDS GTEKPGLVAP ESPVRKSPSE YKLEGRSVSCLKPIEGTLDI 1741 ALLSGPQASK TELPSPESAQ SPSPSGDVRA SVPPVLPSSS GKKNDTTSARELSPSSLKMN 1801 KSYLLEPWFL PPSRGLQNSP AVSLPDPEFK RDRKGPHPTA RSPGTVMESNPQQREGSSPK 1861 HQDHTTDPKL LTCLGQNLHS PDLARPRCPL PPEASPSREK PGLRESSERGPPTARSERSA 1921 ARADTCREPS MELCFPETAK TSDNSKNLLS VGRTHPDFYT QTQAMEKAWAPGGKTNHKDG 1981 PGEARPPPRD NSSLHSAGI P CEKELGKVRR GVEPKPEALL ARRSLQPPGIESEKSEKLSS 2041 FPSLQKDGAK EPERKEQPLQ RHPSSIPPPP LTAKDLSSPA ARQHCSSPSHASGREPGAKP 2101 STAEPSSSPQ DPPKPVAAHS ESSSHKPRPG PDPGPPKTKH PDRSLSSQKPSVGATKGKEP 2161 ATQSLGGSSR EGKGHSKSGP DVFPATPGSQ NKASDGIGQG EGGPSVPLHTDRAPLDAKPQ 2221PTSGGRPLEV LEKPVHLPRP GHPGPSEPAD QKLSAVGEKQ TLSPKHPKPS TVKDCPTLCK 2281QTDNRQTDKS PSQPAANTDR RAEGKKCTEA LYAPAEGDKL EAGLSFVHSE NRLKGAERPA 2341AGVGKGFPEA RGKGPGPQKP PTEADKPNGM KRSPSATGQS SFRSTALPEK SLSCSSSFPE 2401TRAGVREASA ASSDTSSAKA AGGMLELPAP SNRDHRKAQP AGEGRTHMTK SDSLPSFRVS 2461TLPLESHHPD PNTMGGASHR DRALSVTATV GETKGKDPAP AQPPPARKQN VGRDVTKPSP 2521APNTDRPISL SNEKDFVVRQ RRGKESLRSS PHKKAL(SEQ ID NO:9),

[0099] Isotype 10 - NP_001380455.1 1MDESSILRRR GLQKELSLPR RGSLIDSQKW NCLVKRCRTS NRKSLIGNGQ SPALPRPHSP 61LSAHAGNSPQ DSPRNFSPSA SAHFSFARRT DGRRWSLASL PSSGYGTNTP SSTVSSSCSS 121QEKLHQLPYQ PTPDELHFLS KHFCTTESIA TENRCRNTPM RPRSRSLSPG RSPACCDHEI 181IMMNHVYKER FPKATAQMEE RLKEIITSYS PDNVLPLADG VLSFTHHQII ELARDCLDKS 241HQGLITSRYF LELQHKLDKL LQEAHDRSES GELAFIKQLV RKILIVIARP ARLLECLEFD 301PEEFYYLLEA AEGHAKEGQG IKTDIPRYII SQLGLNKDPL EEMAHLGNYD SGTAETPETD 361ESVSSSNASL KLRRKPRESD FETIKLISNG AYGAVYFVRH KESRQRFAMK KINKQNLILR 421NQIQQAFVER DILTFAENPF VVSMYCSFET RRHLCMVMEY VEGGDCATLM KNMGPLPVDM 481ARMYFAETVL ALEYLHNYGI VHRDLKPDNL LVTSMGHIKL TDFGLSKVGL MSMTTNLYEG 541HIEKDAREFL DKQVCGTPEY IAPEVILRQG YGKPVDWWAM GIILYEFLVG CVPFFGDTPE 601ELFGQVISDE INWPEKDEAP PPDAQDLITL LLRQNPLERL GTGGAYEVKQ HRFFRSLDWN 661SLLRQKAEFIPQLESEDDTS YFDTRSEKYH HMETEEEDDT NDEDFNVEIR QFSSCSHRFS 721KVFSSIDRIT QNSAEEKEDS VDKTKSTTLP STETLSWSSE YSEMQQLSTS NSSDTESNRH 781KLSSGLLPKL AISTEGEQDE AASCPGDPHE EPGKPALPPE ECAQEEPEVT TPASTISSST 841LSDMFAVSPL GSPMSPHSLSSDPSSSRDSS PSRDSSAASA SPHQPIVIHS SGKNYGFTIR 901AIRVYVGDSD IYTVHHIVWN VEEGSPACQA GLKAGDLITH INGEPVHGLV HTEVIELLLK 961SGNKVSITTT PFENTSIKTG PARRNSYKSR MVRRSKKSKK KESLERRRSL FKKLAKQPSP 1021LLHTSRSFSC LNRSLSSGES LPGSPTHSLS PRSPTPSYRS TPDFPSGTNS SQSSSPSSSA 1081PNSPAGSGHI RPSTLHGLAP KLGGQRYRSG RRKSAGNIPL SPLARTPSPT PQPTSPQRSP 1141SPLLGHSLGN SKIAQAFPSK MHSPPTIVRH IVRPKSAEPP RSPLLKRVQS EEKLSPSYGS 1201DKKHLCSRKH SLEVTQEEVQ REQSQREAPL QSLDENVCDV PPLSRARPVE QGCLKRPVSR 1261KVGRQESVDD LDRDKLKAKV VVKKADGFPE KQESHQKSHG PGSDLENFAL FKLEEREKKV 1321YPKAVERSST FENKASMQEA PPLGSLLKDA LHKQASVRAS EGAMSDGRVP AEHRQGGGDF 1381RRAPAPGTLQ DGLCHSLDRG ISGKGEGTEK SSQAKELLRC EKLDSKLANIDYLRKKMSLE 1441DKEDNLCPVL KPKMTAGSHE CLPGNPVRPT GGQQEPPPAS ESRAFVSSTH AAQMSAVSFV 1501PLKALTGRVD SGTEKPGLVA PESPVRKSPS EYKLEGRSVS CLKPIEGTLD IALLSGPQAS 1561KTELPSPESA QSPSPSGDVR ASVPPVLPSS SGKKNDTTSA RELSPSSLKM NKSYLLEPWF 1621LPPSRGLQNS PAVSLPDPEF KRDRKGPHPT ARSPGTVMES NPQQREGSSP KHQDHTTDPK 1681LLTCLGQNLH SPDLARPRCP LPPEASPSRE KPGLRESSER GPPTARSERS AARADTCREP 1741SMELCFPETA KTSDNSKNLL SVGRTHPDFY TQTQAMEKAW APGGKTNHKD GPGEARPPPR 1801DNSSLHSAGIPCEKELGKVR RGVEPKPEAL LARRSLQPPG IESEKSEKLS SFPSLQKDGA 1861KEPERKEQPL QRHPSSIPPP PLTAKDLSSP AARQHCSSPS HASGREPGAK PSTAEPSSSP 1921QDPPKPVAAH SESSSHKPRP GPDPGPPKTK HPDRSLSSQK PSVGATKGKE PATQSLGGSS 1981REGKGHSKSG PDVFPATPGS QNKASDGIGQ GEGGPSVPLH TDRAPLDAKP QPTSGGRPLE 2041VLEKPVHLPR PGHPGPSEPA DQKLSAVGEK QTLSPKHPKP STVKDCPTLC KQTDNRQTDK 2101SPSQPAANTD RRAEGKKCTE ALYAPAEGDK LEAGLSFVHS ENRLKGAERP AAGVGKGFPE 2161ARGKGPGPQK PPTEADKPNG MKRSPSATGQ SSFRSTALPE KSLSCSSSFP ETRAGVREAS 2221AASSDTSSAK AAGGMLELPA PSNRDHRKAQ PAGEGRTHMT KSDSLPSFRV STLPLESHHP 2281DPNTMGGASH RDRALSVTAT VGETKGKDPA PAQPPPARKQ NVGRDVTKPS PAPNTDRPIS 2341LSNEKDFVVR QRRGKESLRS SPHKKAL(SEQ ID NO:10),

[0100] Isotype 11 - NP_001380456.1 1MDMSDPNFWT VLSNFTLPHL RSGNRLRRTQ SCRTSNRKSL IGNGQSPALP RPHSPLSAHA 61GNSPQDSPRN FSPSASAHFS FARRTDGRRW SLASLPSSGY GTNTPSSTVS SSCSSQEKLH 121QLPYQPTPDE LHFLSKHFCT TESIATENRC RNTPMRPRSR SLSPGRSPAC CDHEIIMMNH 181VYKERFPKAT AQMEERLKEI ITSYSPDNVL PLADGVLSFT HHQIIELARD CLDKSHQGLI 241TSRYFLELQH KLDKLLQEAH DRSESGELAF IKQLVRKILI VIARPARLLE CLEFDPEEFY 301YLLEAAEGHA KEGQGIKTDIPRYIISQLGL NKDPLEEMAH LGNYDSGTAE TPETDESVSS 361SNASLKLRRK PRESDFETIK LISNGAYGAV YFVRHKESRQ RFAMKKINKQ NLILRNQIQQ 421AFVERDILTF AENPFVVSMY CSFETRRHLC MVMEYVEGGD CATLMKNMGP LPVDMARMYF 481AETVLALEYL HNYGIVHRDL KPDNLLVTSM GHIKLTDFGL SKVGLMSMTT NLYEGHIEKD 541AREFLDKQVC GTPEYIAPEV ILRQGYGKPV DWWAMGIILY EFLVGCVPFF GDTPEELFGQ 601VISDEINWPE KDEAPPPDAQ DLITLLLRQN PLERLGTGGA YEVKQHRFFR SLDWNSLLRQ 661KAEFIPQLES EDDTSYFDTR SEKYHHMETE EEDDTNDEDF NVEIRQFSSC SHRFSKVFSS 721IDRITQNSAE EKEDSVDKTK STTLPSTETL SWSSEYSEMQ QLSTSNSSDT ESNRHKLSSG 781LLPCLAISTE GEQDEAASCP GDPHEEPGKP ALPPEECAQE EPEVTTPAST ISSSTLSDMF 841AVSPLGSPMS PHSLSSDPSS SRDSSPSRDS SAASASPHQP IVIHSSGKNY GFTIRAIRVY 901VGDSDIYTVH HIVWNVEEGS PACQAGLKAG DLITHINGEP VHGLVHTEVI ELLLKSGNKV 961SITTTPFENT SIKTGPARRN SYKSRMVRRS KKSKKKESLE RRRSLFKKLA KQPSPLLHTS 1021RSFSCLNRSL SSGESLPGSP THSLSPRSPT PSYRSTPDFP SGTNSSQSSS PSSSAPNSPA 1081GSGHIRPSTL HGLAPKLGGQ RYRSGRRKSA GNIPLSPLAR TPSPTPQPTS PQRSPSPLLG 1141HSLGNSKIAQ AFPSKMHSPP TIVRHIVRPK SAEPPRSPLL KRVQSEEKLS PSYGSDKKHL 1201CSRKHSLEVT QEEVQREQSQ REAPLQSLDE NVCDVPPLSR ARPVEQGCLK RPVSRKVGRQ 1261ESVDDLDRDK LKAKVVVKKA DGFPEKQESH QKSHGPGSDL ENFALFKLEE REKKVYPKAV 1321ERSSTFENKA SMQEAPPLGS LLKDALHKQA SVRASEGAMSDGRVPAEHRQ GGGDFRRAPA 1381PGTLQDGLCH SLDRGISGKG EGTEKSSQAK ELLRCEKLDS KLANIDYLRK KMSLEDKEDN 1441LCPVLKPKMT AGSHECLPGN PVRPTGGQQE PPPASESRAF VSSTAAQMS AVSFVPLKAL 1501TGRVDSGTEK PGLVAPESPV RKSPSEYKLE GRSVSCLKPI EGTLDIALLS GPQASKTELP 1561SPESAQSPSP SGDVRASVPP VLPSSSGKKN DTTSARELSP SSLKMNKSYL LEPWFLPPSR 1621GLQNSPAVSL PDPEFKRDRK GPHPTARSPG TVMESNPQQR EGSSPKHQDH TTDPKLLTCL 1681GQNLHSPDLA RPRCPLPPEA SPSREKPGLR ESSERGPPTA RSERSAARAD TCREPSMELC 1741FPETAKTSDN SKNLLSVGRT HPDFYTQTQA MEKAWAPGGK TNHKDGPGEA RPPPRDNSSL 1801HSAGIPCEKE LGKVRRGVEP KPEALLARRS LQPPGIESEK SEKLSSFPSL QKDGAKEPER 1861KEQPLQRHPS SIPPPPLTAK DLSSPAARQH CSSPSHASGR EPGAKPSTAE PSSSPQDPPK 1921PVAAHSESSS HKPRPGPDPG PPKTKHPDRS LSSQKPSVGA TKGKEPATQS LGGSSREGKG 1981HSKSGPDVFP ATPGSQNKASDGIGQGEGGPSVPLHTDRAP LDAKPQPTSG GRPLEVLEKP 2041VHLPRPGHPGPSEPADQKLS AVGEKQTLSP KHPKPSTVKD CPTLCKQTDN RQTDKSPSQP 2101AANTDRRAEG KKCTEALYAP AEGDKLEAGL SFVHSENRLK GAERPAAGVG KGFPEARGKG 2161PGPQKPPTEA DKPNGMKRSP SATGQSSFRS TALPEKSLSC SSSFPETRAG VREASAASSD 2221TSSAKAAGGM LELPAPSNRD HRKAQPAGEG RTHMTKSDSL PSFRVSTLPL ESHHPDPNTM 2281GGASHRDRAL SVTATVGETK GKDPAPAQPP PARKQNVGRD VTKPSPAPNT DRPISLSNEK 2341DFVVRQRRGK ESLRSSPHKK AL(SEQ ID NO:11), or

[0101] isotype 12 - NP_001380457.1 1MDESSILRRR GLQKELSLPR RGSFCRTSNR KSLIGNGQSP ALPRPHSPLS AHAGNSPQDS 61PRNFSPSASA HFSFARRTDG RRWSLASLPS SGYGTNTPSS TVSSSCSSQE KLHQLPYQPT 121PDELHFLSKH FCTTESIATE NRCRNTPMRP RSRSLSPGRS PACCDHEIIM MNHVYKERFP 181KATAQMEERL KEIITSYSPD NVLPLADGVL SFTHHQIIEL ARDCLDKSHQ GLITSRYFLE 241 LQHKLDKLLQ EAHDRSESGE LAFIKQLVRK ILIVIARPAR LLECLEFDPE EFYYLLEAAE 301 GHAKEGQGIK TDIPRYIISQ LGLNKDPLEE MAHLGNYDSG TAETPETDES VSSSNASLKL 361 RRKPRESDFE TIKLISNGAY GAVYFVRHKE SRQRFAMKKI NKQNLILRNQ IQQAFVERDI 421 LTFAENPFVV SMYCSFETRR HLCMVMEYVE GGDCATLMKN MGPLPVDMAR MYFAETVLAL 481 EYLHNYGIVH RDLKPDNLLV TSMGHIKLTD FGLSKVGLMS MTTNLYEGHI EKDAREFLDK 541 QVCGTPEYIA PEVILRQGYG KPVDWWAMGI ILYEFLVGCV PFFGDTPEEL FGQVISDEIN 601 WPEKDEAPPP DAQDLITLLL RQNPLERLGT GGAYEVKQHR FFRSLDWNSL LRQKAEFIPQ 661 LESEDDTSYF DTRSEKYHHM ETEEEDDTND EDFNVE IRQF SSCSHRFSKVFSSIDRITQN 721 SAEEKEDSVD KTKSTTLPST ETLSWSSEYS EMQQLSTSNS SDTESNRHKL SSGLLPKLAI 781 STEGEQDEAA SCPGDPHEEP GKPALPPEEC AQEEPEVTTP ASTISSSTLS DMFAVSPLGS 841 PMSPHSLSSD PSSSRDSSPS RDSSAASASP HQPIVIHSSG KNYGFTIRAI RVYVGDSDIY 901 TVHHIVWNVE EGSPACQAGL KAGDLITHIN GEPVHGLVHT EVIELLLKSG NKVSITTTPF 961 ENTS IKTGPA RRNSYKSRMV RRSKKSKKKE SLERRRSLFK KLAKQPS PLLHTSRSFSCLN 1021 RSLSSGESLP GSPTHSLSPR SPTPSYRSTP DFPSGTNSSQ SSSPSSSAPNSPAGSGHIRP 1081 STLHGLAPKL GGQRYRSGRR KSAGNI PLSP LARTPSPTPQ PTSPQRSPSPLLGHSLGNSK 1141 IAQAFPSKMH SPPTIVRHIV RPKSAEPPRS PLLKRVQSEE KLSPSYGSDKKHLCSRKHSL 1201 EVTQEEVQRE QSQREAPLQS LDENVCDVPP LSRARPVEQG CLKRPVSRKVGRQESVDDLD 1261 RDKLKAKVVV KKADGFPEKQ ESHQKSHGPG SDLENFALFK LEEREKKVYPKAVERSSTFE 1321 NKASMQEAPP LGSLLKDALH KQASVRASEG AMSDGRVPAE HRQGGGDFRRAPAPGTLQDG 1381 LCHSLDRGI S GKGEGTEKSS QAKELLRCEK LDSKLANI DY LRKKMSLE DKEDNLCPVLKP 1441 KMTAGSHECL PGNPVRPTGG QQEPPPASES RAFVSSTHAA QMSAVSFVPLKALTGRVDSG 1501 TEKPGLVAPE SPVRKSPSEY KLEGRSVSCL KPIEGTLDIA LLSGPQASKTELPSPESAQS 1561 PSPSGDVRAS VPPVLPSSSG KKNDTTSARE LSPSSLKMNK SYLLEPWFLPPSRGLQNSPA 1621 VSLPDPEFKR DRKGPHPTAR SPGTVMESNP QQREGSSPKH QDHTTDPKLLTCLGQNLHSP 1681 DLARPRCPLP PEASPSREKP GLRESSERGP PTARSERSAA RADTCREPSMELCFPETAKT 1741 SDNSKNLLSV GRTHPDFYTQ TQAMEKAWAP GGKTNHKDGP GEARPPPRDNSSLHSAGIPC 1801 EKELGKVRRG VEPKPEALLA RRSLQPPGIE SEKSEKLSSF PSLQKDGAKEPERKEQPLQR 1861 HPSSIPPPPL TAKDLSSPAA RQHCSSPSHA SGREPGAKPS TAEPSSSPQDPPKPVAAHSE 1921 SSSHKPRPGP DPGPPKTKHP DRSLSSQKPS VGATKGKEPA TQSLGGSSREGKGHSKSGPD 1981 VFPATPGSQN KASDGIGQGE GGPSVPLHTD RAPLDAKPQP TSGGRPLEVLEKPVHLPRPG 2041 HPGPSEPADQ KLSAVGEKQT LSPKHPKPST VKDCPTLCKQ TDNRQTDKSPSQPAANTDRR 2101AEGKKCTEAL YAPAEGDKLE AGLSFVHSEN RLKGAERPAA GVGKGFPEAR GKGPGPQKPP 2161TEADKPNGMK RSPSATGQSS FRSTALPEKS LSCSSSFPET RAGVREASAA SSDTSSAKAA 2221GGMLELPAPS NRDHRKAQPA GEGRTHMTKSSDSLPSFRVST LPLESHHPDP NTMGGASHRD 2281RALSVTATVG ETKGKDPAPA QPPPARKQNV GRDVTKPSPA PNTDRPISLS NEKDFVVRQR 2341RGKESLRSSP HKKAL(SEQ ID NO:12)

[0102] An amino acid sequence or polynucleotide sequence having biologically equivalent activity, even if it is different from the amino acid sequence of SEQ ID NOs: 1 to 12, can also be regarded as the MAST4 protein or its mRNA.

[0103] Therefore, in a specific embodiment, the MAST4 protein may include any one of SEQ ID NOs: 1 to 12 and nucleotide sequences encoding the MAST4 protein.

[0104] The MAST4 protein or polypeptide may include an amino acid sequence having 60% or more, e.g., 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with SEQ ID NOs: 1 to 12. In addition, the MAST4 protein may have an amino acid sequence having modifications of 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, or 7 or more amino acids in the amino acid sequence of SEQ ID NOs: 1 to 12.

[0105] Each polynucleotide encoding MAST4 may have a sequence having 60% or more, such as 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with a sequence encoding any one of the MAST4 proteins. In addition, the polynucleotide encoding MAST4 may be a polynucleotide having a different sequence of 1 or more nucleotides, 2 or more nucleotides, 3 or more nucleotides, 4 or more nucleotides, 5 or more nucleotides, 6 or more nucleotides, or 7 or more nucleotides in the sequence encoding SEQ ID NOs: 1 to 12.

[0106] The present inventors have demonstrated for the first time that bone production can be increased by increasing the expression of the MAST4 gene or stabilizing the expression of the MAST4 gene in eukaryotic cells (eg, mesenchymal stem cells or osteoprogenitor cells).

[0107] Furthermore, in specific embodiments, the composition for promoting the production of extracellular matrix from eukaryotic cells can be used for tissue regeneration or anti-aging.

[0108] The term "pharmaceutically acceptable salt" refers to any organic or inorganic addition salt of a compound in the disclosed composition, which has an effective effect at a concentration that is relatively non-toxic and harmless to the patient, and whose side effects do not reduce the beneficial effects of the disclosed composition. These salts can be selected from any one known to those skilled in the art.

[0109] Compositions of the present disclosure may also include a pharmaceutically acceptable carrier. Compositions including a pharmaceutically acceptable carrier may have various preparations for parenteral administration. When formulated, conventional diluents or excipients (e.g., fillers, extenders, adhesives, wetting agents, disintegrants, surfactants, etc.) may be used to prepare the composition.

[0110] Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), etc. As the base of suppositories, witepsol, macrogol, Tween 61, cocoa butter, lauric acid, glycerin, gelatin, etc. may be used.

[0111] As used herein, "stabilizing MAST4" means preventing degradation of MAST4 or preventing inhibition of the activity of MAST4. For example, MAST4 can be destabilized or degraded by ubiquitination and proteolysis via the proteasome. Therefore, the present invention contemplates inhibitors of agents that target MAST4 for degradation.

[0112] Inhibition of GSK-3

[0113] Glycogen synthase kinase-3 (GSK-3) is a proline-directed serine-threonine kinase. There are two isoforms (GSK-3α and β), which are highly related and largely redundant. Their many substrates range from regulators of cellular metabolism to molecules that control growth and differentiation. Samples of GSK-3β inhibitors include, but are not limited to, Laduviglusib (CHIR-99021), HCl (CAS No.: 1797989-42-4), SB216763 (CAS No.: 280744-09-4), AT7519 (CAS No.: 844442-38-2), CHIR-98014 (CAS No.: 252935-94-7), TWS119 (CAS No.: 601514-19-6), which are some compounds that can be used to inhibit GSK-3β activity. See Selleck Chemicals, Houston, TX (2023). There are also antibodies that specifically inhibit GSK-3β or GSK-3α.

[0114] Therapies for bone building

[0115] The present invention discloses an ex vivo technique involving culturing eukaryotic cells in which a protein that inhibits the production or activity of MAST4 is inhibited from being expressed or inhibited post-translationally, and then transplanting the modified eukaryotic cells into a target bone defect area of ​​a mammalian host, thereby affecting bone formation. Alternatively or simultaneously, resulting in increased expression of MAST4 in the cells.

[0116] One of ordinary skill will appreciate that the preferred source of cells for treating a human patient is the patient's own connective tissue cells or mesenchymal stem cells, such as autologous fibroblasts or osteoblast progenitor cells (osteoprogenitors), osteocytes, pre-osteoblasts, osteoblasts or osteoclasts, but allogeneic cells may also be used.

[0117] More specifically, the method may include the use of an inhibitor of GSK-3, including GSK-3α or GSK-3β.

[0118] Another embodiment of the present invention provides a compound for parenteral administration to a patient in a prophylactically effective amount, the compound comprising a modified cell and a suitable pharmaceutical carrier.

[0119] In the present application, a method is provided for generating or regenerating bone by injecting appropriate mammalian cells, wherein the mammalian cells are transfected or transduced with an overexpressed gene encoding MAST4.

[0120] In embodiments of the present invention, it will be appreciated that cells may be injected into an area where bone is to be generated or regenerated, with or without a scaffold material or any other auxiliary material, such as foreign cells or other biocompatible carriers.

[0121] The methods of the present invention can be applied to all types of bones in the body, including but not limited to non-union fractures (fractures that cannot heal), craniofacial reconstruction, segmental defects due to tumor removal, augmentation of surrounding bone for hip implant correction (i.e., since the lifespan of hip implants is only about 10 years, 25% of hip implants are replacements for existing implants), and jaw reconstruction for dental purposes. Other target bones include vertebrae on the spine for spinal fusion, large bones, etc. In addition, the present invention can be used to treat fractures or defects in the femur, tibia, hip joint, hip fractures, etc. (especially in the elderly) by administering the cells of the present invention to subjects in need.

[0122] The cells to be modified include any suitable mammalian cells, including mesenchymal stem cells and connective tissue cells that contribute to bone formation, including but not limited to fibroblasts, osteogenic progenitor cells, pre-osteoblasts, osteoblasts, osteocytes and osteoclasts, and may also include chondrocytes. However, it should be understood that other non-genetically modified cells may also be included in the composition for contacting the bone defect site, such as pre-osteoblasts, osteoblasts, osteocytes, osteoclasts, chondrocytes, etc.

[0123] When reference is made to "bone defects" or "defective bone," it is understood that such defects may include cracks, breaks, and / or degeneration of bone, including such conditions caused by injury or disease, and may also include defects in the vertebrae of the spine and further degeneration of the disc areas between the vertebrae. In one aspect of the invention, pain caused by degeneration of the disc space between vertebrae may be treated by fusing the vertebrae surrounding the disc space that has degenerated.

[0124] An in vitro method for treating a ruptured or defective bone disclosed in this specification includes initially generating a recombinant virus or plasmid vector containing a DNA sequence encoding a protein or a biologically active fragment thereof. The recombinant vector is then used to infect or transfect an in vitro cultured cell population, thereby producing a cell population containing the vector. These cells are then transplanted into a target bone defect region of a mammalian host, thereby affecting the subsequent expression of the protein or protein fragment in the defect region. The expression of this DNA sequence of interest is useful in substantially repairing the rupture or defect.

[0125] More specifically, the method comprises using a gene encoding MAST4, or a biologically active derivative or fragment thereof.

[0126] Another embodiment of the present invention provides a method for introducing at least one gene encoding a product into at least one cell for treating a mammalian host. The method includes introducing the gene encoding the product into the cell using viral or non-viral means. More specifically, the method includes liposome encapsulation, calcium phosphate coprecipitation, electroporation or DEAE-dextran mediation, and includes using a gene that can encode a MAST4 family member or a biologically active derivative or fragment thereof, and a selective marker or a biologically active derivative or fragment thereof as a gene.

[0127] Another embodiment of the present invention provides for introducing at least one gene encoding a product into at least one cell for use in an additional method for treating a mammalian host. This additional method includes using a biological means of using a virus to deliver a DNA vector molecule to a target cell or tissue. Preferably, the virus is a pseudovirus, and its genome has been altered so that the pseudovirus can only be delivered and stably maintained in the target cell, preferably without retaining the ability to replicate in the target cell or tissue. The altered viral genome is further manipulated by recombinant DNA technology so that the viral genome acts as a DNA vector molecule, and the viral genome comprises a heterologous gene of interest to be expressed in the target cell or tissue.

[0128] A preferred embodiment of the present invention is a method for delivering cells expressing MAST4 protein to a target defect area, wherein the method delivers the MAST4 gene to the tissue of a mammalian host by using an adeno-associated virus vector or a lentivirus vector and the in vitro technique disclosed in this specification. In other words, the DNA sequence of interest encoding a functional MAST4 protein or protein fragment is subcloned into a selected viral vector, and then the recombinant viral vector is cultured to a sufficient titer and used to infect cells cultured in vitro, and the transduced cells are transplanted into the bone defect area or a nearby area where treatment is effective.

[0129] Another preferred method of the present invention involves delivering the MAST4 gene directly to the connective tissue of a mammalian host in vivo by using an adenoviral vector, an adeno-associated virus (AAV) vector or a herpes simplex virus (HSV) vector. In other words, the DNA sequence of interest encoding a functional MAST4 protein or protein fragment is subcloned into a corresponding viral vector. The viral vector containing MAST4 is then cultured to a sufficient titer and directly delivered to the bone defect area or a nearby area where osteogenesis is effective.

[0130] Therapies used to treat osteoporotic fractures

[0131] Osteoporosis is the degeneration of bone structure caused by bone loss caused by imbalance in bone formation, bone resorption or both, so that resorption dominates the bone formation stage, thereby reducing the load-bearing capacity of the affected bones. In healthy adults, the speed of bone formation and resorption is closely coordinated to maintain the renewal of bones. However, in individuals with osteoporosis, the imbalance of these bone remodeling cycles can develop, thereby causing the formation of microstructural defects in bone loss and the continuity of bones. These bone defects produced by small changes (perturbation) in the remodeling sequence can accumulate and eventually reach a point at which the structural integrity of the bones is seriously damaged and may cause fractures. Although this imbalance gradually occurs in most people with age ("senile osteoporosis"), it is much more serious and has a high incidence in postmenopausal women. In addition, osteoporosis can also be caused by nutrition and endocrine disorders, hereditary diseases and some malignant transformations.

[0132] Method for producing cartilage or bone

[0133] Although mesenchymal stem cells have been intensively studied for use in regenerative therapies, the molecular mechanisms that control mesenchymal stem cell differentiation are not fully understood. Our results indicate that Mast4 is a key molecule that determines the commitment and differentiation of MSCs toward either a chondrogenic or osteogenic fate. We have demonstrated that TGF-β1-mediated inhibition of Mast4 gene transcription leads to increased Sox9 protein and Smad3-Sox9 association, resulting in increased Sox9 transcriptional activity, ultimately priming MSCs to promote chondrogenesis at the expense of bone formation. In terms of osteogenesis, we have shown that Wnt-mediated inhibition of Mast4 protein degradation through inhibition of GSK-3β activity leads to increased β-catenin and Runx2 transcriptional activity, ultimately priming MSCs to favor osteogenesis ( Fig.37 ).

[0134] The contextual relevance of TGF-β has been delineated over decades. In particular, the cytostatic effects of TGF-β have been shown to be coordinated by transcriptional activation by CDK inhibitors and repression of c-Myc, highlighting its role in cancer therapy. 31 Many studies have also identified a function for TGF-β in determining the fate of pluripotent stem cells during development. Regarding endochondral ossification during skeletal development, TGF-β promotes mesenchymal condensation and chondrogenesis, but inhibits chondrocyte maturation and differentiation into osteocytes, suggesting that it regulates the order of specific lineages. 32 Observations in animal models support the bifunctionality of TGF-β signaling during skeletal development 15,33We observed inhibition of Mast4 by TGF-β during chondrogenesis in vitro and predominant expression of Mast4 in hypertrophic chondrocytes in vivo. These observations speculate that TGF-β exerts different regulatory effects on skeletogenesis through specific regulation of Mast4 at different stages. Furthermore, our observation that Mast4 had no effect on the cytostatic effects of TGF-β provides compelling evidence supporting that Mast4 is a key mediator of TGF-β-induced chondrogenic differentiation of MSCs.

[0135] Various transcriptional cofactors act as Smad partners to assist in target gene recognition and transcriptional regulation 34 Regarding Smad-mediated gene repression, Smad3 inhibits Runx2 activity through direct interaction, thereby ultimately reducing osteoblast differentiation 35 E2F4 / 5 have been shown to be co-repressors of TGF-β-induced c-Myc repression 36 TGF-β-induced SpB inhibition is associated with the interaction of Smad3 and Nkx2.1 37 Interestingly, analysis of the Mast4 promoter region identified expected binding sites for E2F4 and Nkx2.1 in the vicinity of the Smad3 binding site. Therefore, it will be important to investigate whether these co-transcription factors are involved in TGF-β / Smad3-mediated regulation of Mast4. Furthermore, the discovery of novel co-transcription factors that regulate Mast4 expression during sequential stages of chondrogenic / osteogenic differentiation will contribute to the understanding of cartilage and bone development and their regulation.

[0136] Post-translational modifications (PTMs) regulate protein function and stability and fine-tune signal transduction 38 Here, we demonstrate the impact of PTMs on the TGF-β1-Mast4-Sox9 axis during chondrogenesis. Many signaling pathways and PTMs have been shown to regulate Sox9, a master transcription factor during chondrocyte differentiation, by controlling the cartilage-related ECM gene pool at an early stage. 10 ,16,39 ,40 . Our observations suggest that Mast4 promotes Sox9 degradation by inducing Sox9 phosphorylation at serine 494. Although it remains to be elucidated whether Mast4-induced Sox9 phosphorylation is recognized by either of the E3 ligases for subsequent Sox9 and Mast4 degradation, our studies suggest that Mast4 may be an important factor in controlling Sox9 activity. E6-AP / UBEA is an E3 ligase that induces ubiquitin-mediated proteasomal degradation of Sox9 in hypertrophic chondrocytes during endochondral ossification 41Considering that Mast4 is mainly expressed in hypertrophic chondrocytes, it may be worthwhile to examine whether Mast4 cooperates with E6-AP / UBEA to regulate Sox9 stability in hypertrophic chondrocytes. In addition, it will be necessary to examine the regulation of Sox9 stability by Mast4 through phosphorylation at serine 494 in vivo and the subsequent chondrogenic differentiation capacity in future studies. In addition, previous studies have shown that a delicate balance of Sox9 activity in MSCs is important for proper differentiation. - / - The accumulation of chondrocytes in a mostly terminally differentiated hypertrophic state and their delayed exit from the growth plate shown in the endochondral skeleton of mice can be explained by Mast4-deficiency-mediated overexpression of Sox9 and collagen and reduction of Mmp9 and Mmp13.

[0137] Regarding Wnt / β-catenin, different mechanisms have been reported to explain Wnt-mediated β-catenin stabilization 42 Notably, Wnt inhibits the activity of GSK3 on β-catenin in various ways. Given that phosphorylation by GSK3 generally marks target proteins for ubiquitination and proteolysis, we found that GSK-3β inhibited Mast4 phosphorylation, increased the stability of Mast4, and subsequently β-catenin, enhancing the role of Wnt / β-catenin signaling in MSC selection of osteoblast fate. 24,43 In addition, it would be worthwhile to examine Mast4 protein levels in GSK-3β-deficient mice and mice administered a GSK-3β inhibitor, which show increased bone formation and bone mass. 21 ,44 ,45 .

[0138] Mast4 belongs to the MAST kinase family, which consists of Mast1-4 and Mast1 46 Mast1 to 4 have a similar domain organization with a kinase domain, a PDZ domain, and a domain of unknown function (DUF). Currently, little is known about the biological roles of the MAST kinase family. Several studies have shown that Mast1, 2, and 3 are associated with cancer. 47,48 In addition to its role as a neuroprotective mediator 49-51 , have reported that Mast4 undergoes O-GlcNAc modification, in which an overall increase is often observed during osteoblast differentiation 52 Furthermore, the study showed that Mast4-mediated FGF-2 signaling in Sertoli cells (known to play a role in bone formation) was activated by inducing ERM phosphorylation at serine 367 residue 53 Meanwhile, the microtubule cytoskeleton has been shown to contribute to the osteogenic differentiation of mesenchymal stem cells. 54Since the MAST kinase family shares a high degree of similarity in protein domains that are considered structural and functional building blocks, it is likely that MAST kinase family members are key cellular mediators of various signal transductions in normal and disease states. In addition, examining Mast4 regulation in the differentiation of mesenchymal stem cells into various lineages, including osteoblasts or adipocytes, may warrant further investigation.

[0139] In conclusion, we have demonstrated that Mast4 is a key mediator of MSC commitment to the osteochondrogenic differentiation pathway. Our study implies that Masts4 functions in limiting Sox9 transcriptional activity, thereby determining the fate of MSCs to develop into cartilage or bone. Therefore, in the context of cell therapy, Mast4 would be an ideal target for potential MSC therapy.

[0140] Therapeutic compositions

[0141] The formulation of therapeutic compounds is generally known in the art and can be conveniently referenced to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, about 0.05 μg to about 20 mg per kilogram of body weight can be administered per day. The dosage regimen can be adjusted to provide the best therapeutic response. For example, several separate doses can be administered per day, or the dose can be reduced proportionally, depending on the urgency of the treatment situation. The active compound can be administered in a convenient manner, such as by intravenous (where water soluble), intramuscular, subcutaneous, intranasal or intradermal administration.

[0142] The pharmaceutical form suitable for injectable use includes sterile aqueous solutions (water-soluble therein) or dispersants and sterile powders for the temporary preparation of sterile injectable solutions or dispersants. In all cases, the form must be sterile and must have fluidity to the extent of easy injection. It must remain stable under manufacturing and storage conditions and must prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. For example, by using a coating (such as lecithin), by maintaining the required particle size in the case of dispersion and by using a surfactant, appropriate fluidity can be maintained. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (such as chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be caused by using agents that delay absorption (such as aluminum monostearate and gelatin) in the composition.

[0143] Sterile injectable solutions are prepared by incorporating the required amount of the active compound into an appropriate solvent with the various other ingredients listed above, followed by filtering and sterilizing as needed. Typically, dispersions are prepared by incorporating various sterile active ingredients into a sterile carrier that contains a basic dispersion medium and the necessary other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying techniques that produce a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered solution.

[0144] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unitary dosages for mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active materials for treating disease in living subjects having disease conditions in which physical health is compromised.

[0145] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. The following examples are provided as illustrations of the present invention, rather than limiting the present invention. Example

[0146] All animal studies were approved by the Institutional Animal Care and Use Committee of KNOTUS Co., Ltd and the Center for Phenogenomics Animal Research Facility and Woojung BSC and were performed in accordance with ethical and procedural guidelines. Laboratory mice were maintained at room temperature (20-22°C) and constant humidity (40±10%) with a 12 h light / dark cycle.

[0147] Example 1 - Generation of Mast4 knockout mice using CRISPR / Cas9 technology

[0148] To generate Mast4 knockout mice by CRISPR / Cas9-mediated gene targeting, we targeted exon 1 and exon 15 of Mast4 (reference sequence accession number: 175171): 5'-GGAAACTCTGTCGGAGGAAG-3' (SEQ ID NO: 13) (exon 1) and 5'-GGCACAAAGAGTCCCGCCAG-3' (SEQ ID NO: 14) (exon 15). We then inserted each sequence into the pX330 plasmid carrying the guide RNA and Cas9 expression unit, which was provided by Dr. Feng Zhang (Addgene, #42230). 55 We named these vectors pX330-Mast4-E1 and pX330-Mast4-E15.

[0149] Pregnant mare serum gonadotropin (5 units) and human chorionic gonadotropin (5 units) were injected intraperitoneally into female C57BL / 6J mice (Charles River Laboratories, Kanagawa, Japan) at 48h intervals, and then the female C57BL / 6J mice were mated with male C57BL / 6J mice. PX330-Mast4-E1 and pX330-Mast4-E15 (round, 5ng / μl each) were co-microinjected into 231 fertilized eggs collected from the oviduct of mated female mice. The 225 fertilized eggs that survived the injection were transplanted into the oviduct of pseudo-pregnant ICR females, and 47 newborn mice were obtained. Genomic DNA was collected from the tails of 31 surviving founder mice.

[0150] To confirm CRISPR / Cas9-induced indel mutations, we amplified the genomic region including the target site by PCR using primers for exon 1 target (MAST4-1 genotype F: 5'-GTAGGGACTCCACGCTCCAG-3' (SEQ ID NO: 15); MAST4-1 genotype R: 5'-CCGGACCCTAGTCTCTTCG-3' (SEQ ID NO: 16)) and exon 15 target (MAST4-15 genotype F: 5'-GGGTTCTCTGCGAAAGTCAG-3' (SEQ ID NO: 17); MAST4-15 genotype R: 5'-ATCCCTGTGTTCCGTTTCAG-3' (SEQ ID NO: 18). The genomic region was amplified by PCR using the BigDye Terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific, Inc., MAST4-1500). Scientific), MAST4-1 genotype F primers and MAST4-15 genotype F primers were used to sequence the PCR products. In male founder mouse #38, we found insertion / deletion mutations in both exon 1 and exon 15, without random integration of pX330. In order to identify the insertion / deletion sequence and whether the insertion / deletion mutations of exon 1 and exon 15 occurred on the same chromosome (cis mode), founder mouse #38 was mated with wild-type females, and the insertion / deletion mutations in F1 were sequenced. 17 F1 newborn mice were obtained, of which 12 of them deleted 71bp in exon 1 in cis mode (chr13: 103,333,981-103,334,051: GRCm38 / mm10) and 3bp in exon 15 (chr13: 102,774,360-102,774,362).

[0151] Example 2 - CRISPR / Cas9-mediated depletion of Mast4 in C3H10T1 / 2 and human bone marrow-derived stem cells

[0152] For C3H10T1 / 2 cells, the lentiCRISPRv2 vector (Addgene, #52961) was digested with BsmBI and ligated to annealed oligonucleotides targeting Mast4 exon 1 (5'-TACCCTGCCGCTGCCGCACC-3' (SEQ ID NO: 19)) (LentiCRISPRv2-Mast4Ex1) and exon 2 (5'-AGCAACCCAGATGTGGCCTG-3' (SEQ ID NO: 20)) (LentiCRISPRv2-Mast4Ex2). To generate lentivirus, HEK293T cells were transfected with LentiCRISPRv2-Mast4Ex1 and packaging vectors (pVSVG and psPAX2, Addgene #8454, #12260) at 70% confluence using polyethyleneimine. Viral supernatants were harvested 48 h after transfection, filtered through a 0.45 μm filter and applied to C3H10T1 / 2 cells. After puromycin-mediated selection, single-cell clones were grown in 96-well plates. From genomic DNA, the DNA was isolated using AccuPower TM PCR premix (Bioneer) was used to amplify the exon 1 and exon 2 regions of the Mast4 gene. Indel mutations leading to frameshift-mediated depletion of Mast4 protein were confirmed by sequencing. For human bone marrow-derived stem cells (hBMSCs), we used GeneArt TM Guide RNA (gRNA) was generated by Precision gRNA Synthesis Kit (Invitrogen). At passage 5 to 6, human bone marrow-derived stem cells were transfected with gRNA targeting exon 5 (forward: 5'-TAATACGACTCACTATAGAGCAACCGGAAAAGCTTAAT-3' (SEQ ID NO: 21); reverse: 5'-TTCTAGCTCTAAAACATTAAGCTTTTCCGGTTGCT-3' (SEQ ID NO: 22)) and Cas9 protein (Toolgen) using the Neon transfection system according to the manufacturer's protocol. Since hBMSCs cannot form colonies from single cells, the edited cell pools were used for further chondrogenic differentiation, protein and mRNA isolation. The efficiency of CRISPR / Cas9-mediated Mast4 gene knockout in hBMSCs was determined by ICE knockout analysis (www.synthego.com). Mast4-depleted hBMSCs obtained ICE and KO scores of >70, which indicated the use of the indel percentage and the proportion of cells with frameshifts or 21+bp indels, respectively. Example 3 - Lentiviral shRNA production / infection

[0153] Two different shRNAs targeting exon 15 and exon 22 of Mast4 (shMast4 exon 15F: CCGGCCCAGTTGATATGGCCAGAATCTCGAGATTCTGGCCATATCAACTGGGTT TTTG (SEQ ID NO: 23), shMast4 exon 22F: CCGGCCGAAGTTTCTCCTGCTTAAACTCGAGTTTAAGCAGGAGAAACTTCGGTT TTTG (SEQ ID NO: 24)) were designed, and the annealed oligonucleotides were inserted into the pLKO.1 vector. To generate shRNA lentivirus, 293T cells were transfected with pLKO-shMast4 (#1 and #2) or scrambled control pLKO-pGL2 together with lentiviral packaging plasmids psPax2 and VSV-G. 48 h after transfection, the viral supernatant was harvested and filtered. C3H10T1 / 2 cells were infected with shRNA lentivirus and polybrene (8 μg / ml) for 24 h, followed by puromycin selection (4 μg / ml).

[0154] Example 4 - Cell culture and chondrogenic / osteogenic differentiation

[0155] C3H10T1 / 2 cells (clone 8, CCL-2260, ATCC), mouse bone marrow-derived mesenchymal stromal cells (mBMSCs), and human embryonic kidney cell line HEK293T (CRL-3216, ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM; LM001-05, WELGENE) containing 10% fetal bovine serum (FBS; S001-01, WELGENE) and 1% penicillin-streptomycin (P / S; LS202-02, WELGENE). ATDC5 (RCB0565, RIKEN BRC) cells were cultured in DMEM / F-12 (11320033, Gibco) containing 5% FBS and 1% P / S. hBMSCs were kindly provided by SCM Lifescience (Incheon, South Korea), in which the hBMSC line was established by the subfractionation culture method. 56. Briefly, human bone marrow aspirated from the iliac crest of three healthy donors was mixed with isolation medium and incubated after obtaining written informed consent approved by the Inha University Hospital Institutional Review Board (IRB number 10-51). The supernatant containing floating bone marrow cells (cells that did not settle to the bottom) was repeatedly transferred to new 100 mm culture dishes. After 10 to 14 days of incubation, well-isolated colonies were isolated, expanded, and characterized. These cells were cultured in DMEM (low glucose; LM001-11, WELGENE) containing 10% FBS and 1% P / S. Human primary chondrocytes were also kindly provided by SCM Lifescience, which were collected by staining collagenase-treated cartilage tissue obtained from a 1-year-old human female donor. 57 . These cells were cultured in DMEM (17-205-CVR, Corning) containing 10% FBS (26140-079, Gibco), 20 mM L-glutamine (25030-081, Gibco) and 10 μg / ml gentamicin (15700-060, Thermo fisher). MC3T3-E1 cells (subclone 4, CRL-2593, ATCC) were cultured in α-minimal essential medium (α-MEM) without ascorbic acid (LM008-53, WELGENE) and containing 10% FBS and 1% P / S. All cells were cultured at 37°C in a humidified 5% CO2 incubator. For micromass cultures of C3H10T1 / 2 cells, 1×10 5 10 cells were seeded onto the culture dish, followed by a 2-hour attachment period. Then, medium containing BMP-2 (150 ng / ml; PeproTech) was added to the culture dish, and the medium was changed every 48 to 72 hours. For the pellet culture of hBMSCs, 2×10 5 The cells were inoculated into a 15 ml conical tube and incubated in a 1% P / S, 10 -71 / 100 of ITS+ premixed universal culture supplement (Premix Universal Culture Supplement, Corning), 50ng / ml ascorbic acid (Sigma Aldrich), 10ng / ml of TGF-β1 and TGF-β3 (R&D system) and 40ng / ml of L-proline (Sigma Aldrich) in α-MEM for 21 days. The culture medium was changed every 48 to 72h. For mBMSC, cells were isolated from bone marrow aspirates harvested from the tibial bone marrow compartment and cultured in DMEM containing 10% FBS for 3h. Non-attached cells were carefully removed and fresh culture medium was re-supplied. According to the manufacturer's instructions, the cultured BMMSCs were differentiated into chondrocytes using the StemPro Chondrogenesis Differentiation Kit (StemPro Chondrogenesis Differentiation Kit, Thermo Fisher Scientific). For osteogenic differentiation of C3H10T1 / 2 cells, confluent cells were cultured for 10 days in maintenance medium supplemented with 50 μg / ml ascorbic acid (Sigma Aldrich), 10 mM β-glycerophosphate (Sigma Aldrich), and 200 ng / ml BMP-2. The medium was changed every 48 to 72 h.

[0156] Example 5 - Alcian blue and alkaline phosphatase (ALP) staining

[0157] Differentiated cells were washed twice with PBS and fixed in 4% paraformaldehyde for 5 to 10 minutes at room temperature. Osteogenic cells were stained with alcian blue solution (1% alcian blue in 0.1M HCl solution, pH 1.0; Sigma Aldrich) overnight, then washed once with 0.1M HCl, and washed twice with PBS. Osteogenic cells were stained with 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium solution (BCIP / NBT; Merck) at 37°C for 30 min.

[0158] Example 6 - 3D spheroid formation assay

[0159] 3D spheroid formation of C3H10T1 / 2 cells was performed using low binding plates as previously reported. 23 Briefly, a round-bottom ultra-low attachment 96-well microplate (Corning) was coated with gelatin (0.1%; Sigma Aldrich). Then, 1×10 5Cells were added to each well of the coated microplate and cultured for 8 days. The culture medium was changed every 48 to 72 h.

[0160] Example 7 - RNA-Seq and bioinformatics analysis

[0161] For sample preparation for RNA sequencing using differentiated C3H10T1 / 2 cells, a total of 30 high-density micromass cultures obtained from three separate inductions of chondrogenic differentiation (10 micromass / each induction) of wild-type and Mast4-depleted (KO#1) C3H10T1 / 2 cells were pooled together for RNA sequencing. For RNA sequencing using mouse cartilage and bone, cartilage and bone were dissected as follows. Mast4 was expressed on day 1 of PN in a CO2 chamber. + / + and Mast4 - / - After the mice were euthanized, the middle part of the femur was cut open. After removing the skin, all muscles were removed using forceps. The fibula was removed after cutting the articular cartilage of the knee joint and tibiotalar joint. The epiphysis was separated from the tibial body along the border of the calcified area using a 30G needle. The separated epiphyseal cartilage and tibia were placed in (Invitrogen). Tibiae were cut in half and minced using a razor blade. Each sample was homogenized using an equal amount of 0.5 mm stainless steel beads. RNA was then obtained by layer separation using chloroform and precipitation using isopropanol. Since the total amount of RNA obtained from each mouse on PN day 1 was insufficient for RNA sequencing analysis, the RNA isolated from Mast4 on PN day 1 was combined. + / + and Mast4 - / - RNA was obtained from tibial cartilage or bone of mice (n = 3 per group). 1 μg of qualified RNA was used in each sample, and RNA-Seq libraries were prepared using the TruSeq RNA Sample Preparation Kit according to the manufacturer's manual (Illumina, Inc., San Diego, CA). After qPCR validation, the libraries were paired-end sequenced using the Illumina HiSeq2500 platform with a read length of 100 bp, resulting in an average of 57.7 million reads per library. The quality of raw reads was assessed using FastQC (version 0.11.9). TopHat 58 Clean reads of each sample with an average quality score greater than Q30 were aligned to the mouse reference genome GRCm38.p4mm10 using the gene model annotation group. Gene expression was calculated as FPKM using Cufflinks. 59Differential expression analysis between wild-type and Mast4-depleted samples was performed with a cutoff set at P < 0.05 and ≥ 1.5 fold difference compared to qPCR validation of Sox9-targeted genes. 60 Gene ontology (GO) enrichment analysis was performed on the DEG dataset with a cutoff value of P < 0.001. Interactions of genes related to cartilage and / or bone development, BMP signaling, TGF-β signaling, and Wnt signaling were searched with high confidence (≥ 0.7) using the STRING database (https: / / string-db.org / ) and further analyzed using Cytoscape (www.cytoscape.org) based on the connectivity of the nodes. Gene set enrichment analysis (GSEA) was performed using the background dataset (www.gsea-msigdb.org / gsea / index.jsp) 61 The background data set is from Mast4 at PN 1 day. + / + and Mast4 - / - Composition of all DEGs analyzed in tibial cartilage and bone of mice, Mast4 + / + and Mast4 - / - Mouse expression >0.3 FPKM, which balances Mast4 + / + or Mast4 - / - False positives and false negatives in mice 62 quantity.

[0162] Example 8 - Reverse transcription PCR (RT-PCR) and real-time RT-PCR

[0163] Total RNA was prepared using EasyBlue (Boca Scientific). 2 μg of RNA was reverse transcribed using M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. The PCR products were analyzed using the AccuPower PCR with specific primer pairs. TM RT-PCR was performed using a PCR premix (Bioneer). Quantitative real-time PCR was performed using a PowerSYBR Green PCR premix (Applied Biosystems) on a QuantStudio 5 real-time PCR instrument (Applied Biosystems). The mRNA level of each gene was measured three times and normalized using Gapdh. Information about the oligonucleotides used in this study is as follows.

[0164] Example 9 - Histological Analysis

[0165] For cartilage immunofluorescence staining, the tissue was fixed overnight with 4% paraformaldehyde (Wako) in 0.01M PBS (pH 7.4) at 4°C and then decalcified with 10% EDTA solution. After embedding in paraffin (Leica Biosystems), the samples were sliced ​​at a thickness of 6 μm. The tissue sections were incubated with primary antibodies against Mast4 (Bioworld Technology), Col2a1 (Abcam) and Sox9 (Cell Signaling Technology) at 4°C overnight. After washing in PBS, the tissue sections were continuously incubated in AlexaFluor488 (Invitrogen) for 2 h at room temperature. The TO-PRO TM-3 (Invitrogen) counter-stains tissue sections for 15 minutes. Images were taken using a confocal microscope DMi8 (Leica). In order to detect collagen tissue, freshly prepared Russell-Movat modified five-color staining (MasterTech, USA) was used to stain the sections according to the manufacturer's protocol. Images were made into binary at standard thresholds, and positive pixels were counted using a Leica Microsystems CTR 6000 (Leica). For skeletal immunofluorescence staining, mice were anesthetized and fixed by perfusion with 4% PFA to collect femurs and tibias. Samples were fixed overnight using 2% PFA at 4°C. Samples were decalcified in a 0.5M EDTA solution for 6 days. Then, the samples were embedded in 5% low melting point agarose (Invitrogen) and cut into 150 μm slices by a vibrating slicer (Leica, CT1200S). After removing agarose from the slices, the slices were infiltrated with PBST (0.3% Triton X-100 in phosphate buffered saline) for 20 minutes and blocked with 5% goat serum in PBST for 30 minutes. The slices were incubated at room temperature for 2h with primary antibodies diluted in blocking solution, washed 3 times with PBS, and processed in blocking solution with secondary antibodies for 75 minutes at room temperature. After washing the slices 3 times in PBST and 3 times in PBS, the slices were mounted on microscope slides using fluorescent mounting media (DAKO). The primary antibodies and reagents used for immunofluorescence are as follows: CD31 (Millipore, MAB1398Zm, 1: 150), MMP13 (Abcam, 1: 150), Osterix (Abcam, 1: 300), Runx2 monoclonal antibody (Cell Signaling Technology, 1: 150). Secondary antibodies and reagents used for IF were as follows: FITC-conjugated anti-hamster IgG (Jackson ImmunoResearch, 1:300), Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch, 1:300). Stained bone sections were analyzed at high resolution using a Zeiss LSM 880 confocal microscope (Carl Zeiss). Z-stacks of images were processed using Zen software.

[0166] Example 10 - Element Mapping via EPMA

[0167] Elemental mapping of Ca, P, and Mg was performed using an electron probe microanalyzer (EPMA-1610; Shimadzu, Tokyo, Japan). Undecalcified 6-week-old mouse tibiae were embedded in epoxy resin and trimmed with a diamond disk until the sagittal plane was exposed. After polishing, the samples were sputter-coated with carbon before elemental analysis. For each experiment, 256 × 256 pixel mapping was performed. The accelerating voltage and beam current were set to 15 kV and 0.03 μA, respectively, and the integration time for each pixel was 0.05 s.

[0168] Example 11 - Micro-CT

[0169] Three-dimensional reconstructed computed tomography images were obtained by scanning the calcified SPC-generated bone regions using a MicroCT Skyscan 1076 (Antwerp). The data were then digitized using an image acquisition card, and the resulting images were transferred to a computer for analysis using Comprehensive TeX Archive Network (CTAN) topographic reconstruction software.

[0170] Example 12 - In vivo calcein labeling

[0171] Three-week-old mice were injected intraperitoneally with 50 mg / kg calcein (Sigma-Aldrich, St. Louis, MO) in 5% sodium bicarbonate solution. Mice were labeled 7 and 2 days before sacrifice. Femurs were fixed in 4% formaldehyde at room temperature for 1 day. Samples were incubated in 10% (v / v) KOH for 96 h and embedded in paraffin as previously described. 63 The embedded samples were sliced ​​into 5 μm thickness and observed using a confocal microscope (Leica, DMi8, Germany). The distances between the marks on the cortical bone were measured at 3 points for each sample.

[0172] Example 13 - Growth Plate Morphology Measurements

[0173] For morphometric analysis, the total thickness of the growth plate cartilage at each proximal tibia is measured at H&E staining slice image or five-color staining slice image, which is equidistantly spaced along the axis 90 ° with the transverse plane of the growth plate and parallel to the longitudinal axis of the skeleton. Three measured values ​​are obtained from each epiphyseal growth plate, and the final thickness determination of individual animals is shown to have the mean value of these values ​​using image analysis software (U.S., National Institutes of Health (NIH), ImageJ, version 1.38e). The width of the layer occupied by hypertrophic chondrocytes is measured by the same method. In addition, the percentage of the total thickness of the growth plate accounted for by the mast cell layer is also calculated. Three left and right tibiae are used in each group.

[0174] Example 14 - Isolation of mouse skeletal stem cells using flow cytometry

[0175] Reference Solution 27 Briefly, 5-week-old male Mast4 + / + and Mast4 - / - Mice (n=5) were isolated and the humerus, femur and tibia were dissected. Cells were isolated by a combination of mechanical and chemical digestion, and red blood cells were removed by ammonium-chloride-potassium (ACK) lysis buffer. TER119 was filtered by magnetic activated cell sorting (MACS). + CD45 + Hematopoietic cells. The remaining cells were then stained with the following antibodies: CD45, TER119, TIE2, ITGAV, CD202B, THY1.1, THY1.2, CD105 and 6C3. 7-AAD was used to distinguish live cells / dead cells. FACS analysis was performed on a FACS Aria ll instrument (BD Biosciences) and analyzed by FlowJo v10.7.1 and BD FACSDiva v9.0.1 software.

[0176] Example 15 - In vivo chondrogenesis assay

[0177] Control cells and Mast4-depleted C3H10T1 / 2 cells were cultured in micromass cultures in chondrogenic differentiation medium including BMP-2 (150 ng / ml) for 4 days. Cells were resuspended in PBS (100 micromass cultures in 100 μl per injection) and injected subcutaneously into the flank of athymic nude mice (6-week-old females; n=4). After 2 weeks, mice were euthanized and grafts were collected for IHC evaluation. V was measured and expressed using the formula V = (A*B 2 ) / 2 to calculate the volume of the graft containing cartilage, where V is the volume (mm 3 ), A is the long diameter (mm), and B is the short diameter (mm). All experiments were performed in accordance with the guidelines provided by the Laboratory Animal Care and Use Committee of the Phenogenomics Animal Research Center of Woojung BSC (an accredited institution of the Association for Assessment and Accreditation of Laboratory Animal Care, Suwon, South Korea).

[0178] Example 16 - In vivo transplantation in a full-thickness cartilage defect rabbit model

[0179] As previously reported 64, a full-thickness cartilage defect model was prepared. In brief, 13 healthy New Zealand white male rabbits (weight 3.0-3.5kg) were obtained 4 weeks before the experiment. The rabbits were anesthetized using Zoletil and xylazine. Under sterile conditions, a parapatellar skin incision was made on the right knee, and the patella was dislocated laterally. A full-thickness cartilage defect (3mm in diameter, 3mm in depth) was formed in the center of the trochlear groove by drilling. Cartilage and bone debris were removed, and the defect site was carefully cleaned with saline. The vehicle (PBS 50μl), original or MAST4-depleted hBMSCs (2×10 in 50μl) were added. 6 hBMSCs (n=3 for vehicle, n=5 for naive and MAST4-depleted hBMSCs) were transplanted into the defect site, and then the patella was repositioned. The wound was closed with 4-0 nylon sutures. All procedures were performed in accordance with the guidelines provided by the Laboratory Animal Care and Use Committee of KNOTUS Co., Ltd (Incheon, South Korea).

[0180] Example 17 - Chromatin Immunoprecipitation

[0181] Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. Glycine was added to a final concentration of 125 mM for 5 minutes to quench formaldehyde cross-linking. Cells were washed with ice-cold phosphate-buffered saline, harvested by scraping, precipitated and resuspended in SDS lysis buffer (50 mM Tris-HCl [pH 8.1], 1% SDS, 10 mM EDTA) containing a complete protease inhibitor cocktail (Roche). Cell extracts were sonicated using a Bioruptor TOS-UCW-310-EX (output, 250 W; 23 ultrasonic cycles, 30 second intervals; Cosmo Bio). Samples were centrifuged at 18,472 x g for 10 minutes at 4°C, and the supernatant was diluted 10-fold in dilution buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1% Triton X-100, 150 mM NaCl and a complete protease inhibitor cocktail). Chromatin samples were pre-cleared for 2 h using protein A-agarose beads (Santa Cruz) and then immunoprecipitated overnight at 4°C against Sox9 and Smad3 (Abcam) antibodies. Immune complexes were collected using protein A-agarose beads. Samples were washed five times (the first wash was with low-salt immune complex wash buffer [20 mM Tris-HCl, pH.8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 150 mM NaCl], the second wash was with high-salt immune complex wash buffer [20 mM Tris-HCl, pH.8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 500 mM NaCl], the third wash was with LiCl immune complex wash buffer [10 mM Tris-HCl, pH.8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40 and 1% sodium deoxycholate], and the final wash was twice with TE buffer). Immunoprecipitated samples were eluted at room temperature using a buffer containing 1% SDS and 100 mM NaHCO3. The eluate was heated overnight at 65°C to reverse the crosslinking after adding NaCl to a final concentration of 100 mM. Genomic DNA was extracted using a PCR purification kit (GeneAll). Chromatin was precipitated by real-time PCR and the readings were normalized using 5% input chromatin for each sample. The experiment was repeated two or more times. The binding of Sox 9 to the Col2a1 gene was detected using the forward primer 5'-AACCCTGCCCGTATTTATTT-3' (SEQ ID NO: 25) and the reverse primer 5'-TGTGCATTGTGGGAGAGG-3' (SEQ ID NO: 26).The binding of Smad3 to the Mast4 gene was detected using the forward primer 5'-TGCTGACACTTTATTTTGCTCT-3' (SEQ ID NO: 27) and the reverse primer 5'-CATCTCCAAGCCTCTTTCTG-3' (SEQ ID NO: 28).

[0182] Example 18 - Ubiquitination Assay

[0183] Flag-MAST4-PDZ, GFP-Smruf1, GFP-GSK-3β and HA-Ub plasmids were transfected into C3H10T1 / 2 cells and then treated with MG-132 (10 μM for 6 h). Cells were lysed by boiling for 10 min in SDS lysis buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% SDS, 5 mM NEM, protease inhibitors] and then diluted 10 times using dilution buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100]. Lysed samples were immunoprecipitated overnight using Flag antibody (Sigma-Aldrich), and antibody-bound proteins were precipitated using Dynabeads. The precipitated samples were washed with wash buffer A [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.1% SDS] and B [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100] and then subjected to western blotting.

[0184] Example 19 - Luciferase Assay

[0185] 4xCol2a1-luc, Smad3 / 4-responsive promoter (CAGA) was used by using polyethyleneimine (Polysciences) 12 -luc, SBE-luc, 6xOSE-luc, MAST4-promoter luciferase reporter plasmid, HA-MAST4PDZ, Myc-Sox9WT / S494A / S494D plasmid transient transfection C3H10T1 / 2 cells. TGF-β1 (3ng / ml continues 24h) (R&D Systems) and Vactosertib (500nM continues 26 hours) process cells are used. According to the manufacturer's scheme, luciferase activity is analyzed using luciferase assay system kit (Promega). All determinations are repeated three times to complete, and all values ​​are normalized to measure the transfection efficiency for beta-galactosidase activity.

[0186] Example 20 - Immunoprecipitation Assay and Western Blot Analysis

[0187] For immunoprecipitation assays, cell extracts were incubated with the indicated primary antibodies overnight at 4°C. Antibody-bound proteins were precipitated using Dynabeads Protein G (Invitrogen). Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Complete; Roche). Samples were separated by SDS-PAGE and then electrotransferred to polyvinylidene difluoride membranes (PVDF; Millipore). The membranes were blocked for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated antibodies (Millipore) were used as secondary antibodies. Peroxidase reaction products were visualized using WESTZOL (Intron). All signals were detected by Amersham Imager 600 (GE Healthcare LifeSciences).

[0188] Example 21 - In-gel digestion for sample preparation for mass spectrometry

[0189] Gel bands corresponding to the size of Myc-Sox9 were excised, and 50% (v / v) acetonitrile (ACN) prepared in 25mM ammonium bicarbonate and 100mM ammonium bicarbonate was used to decolorize the gel bands for 15min. Proteins were reduced for 1h using 20mM DTT at 60°C, and then alkylated for 45min using 55mM iodoacetamide at room temperature in the dark. After dehydration, proteins were digested overnight using a mass spectrometry grade trypsin / Lys-C mixture (Promega, Madison, Wisconsin, USA) prepared in 50mM ammonium bicarbonate at 37°C. Peptides were extracted from gel fragments using 50% (v / v) acetonitrile prepared in 5% formic acid, dried under a Centrivap concentrator (Labconco, Kansas City, Missouri, USA), and stored at -20°C until use.

[0190] Example 22 - Mass spectrometry for detection of Sox9 phosphorylation

[0191] Peptide samples extracted by in-gel digestion were suspended in 20 μl of solvent A (0.1% formic acid prepared in water, Optima LC / MS grade, ThermoFisher Scientific). Thereafter, 4 μl of sample was loaded onto an EASYSpray C18 column (75 μm×50 cm, 2 μm) and separated at a flow rate of 300 nL / min for 65 min using a 2 to 35% gradient of solvent B (0.1% formic acid prepared in ACN). Mass spectra were recorded on a Q Exactive hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific), which was interfaced with a nano-ultra HPLC system (Easy-nLC1000; Thermo Scientific). The spray voltage was set to 1.5 kV, and the temperature of the heated capillary was set to 250°C. Q-Exactive was run in data-dependent mode, and each survey cycle consisted of a full MS scan with a mass range of 300-1400 m / z and an MS / MS scan of the 10 most intense ions. The exclusion time for previously fragmented peptides was 20 sec. Peptides were fragmented using high-energy collisional dissociation, and the normalized collision energy value was set to 27%. The resolution for full MS scans and MS / MS scans was 70,000 and 17,500, respectively. The advanced gain control target was 5 × 10 4 , the maximum injection time was 120 ms, and the isolation window was set to 3 m / z.

[0192] Raw data was processed using Trans-Proteomic Pipeline (v4.8.0PHILAE) to convert into a searchable mzXML document. The database search of sequenced peptides was the Sequest (version 27) algorithm in the SORCERER (Sage-N Research, Milpitas) platform with the Uniprot human database. Parent and fragment ion tolerances were set to 10ppm (monoisotope) and 1Da (monoisotope), respectively. 57Da cysteine ​​(carbamidomethylation) was fixedly modified. Various modifications were made to 16Da methionine (oxidation) and 80Da serine, threonine, tyrosine (phosphorylation). Trypsin was selected as an enzyme with a maximum allowable miss of cleavage up to twice. Peptide and protein identification based on MS / MS was verified using the scaffold software package (Proteome Software Inc., Portland, Oregon, USA, version 3.4.9). The thresholds for peptide and protein identification were 95% minimum and 95% minimum, respectively, with a minimum of 2 peptides. The peptide and protein FDRs were 0.2% (decoy) and 0.6% (decoy), respectively.

[0193] Example 23 - Statistical Analysis

[0194] All quantitative experiments were repeated three times and / or repeated at least three times. Data are expressed as mean ± SD. Student's t test was performed using GraphPad Prism version 5 (GraphPad Software Inc.). P < 0.05 was considered statistically significant. Significance was achieved at P < 0.05.

[0195] Example 24 - Results

[0196] Example 24.1 - Mast4 regulates osteochondrogenic gene expression in MSCs

[0197] We found that microtubule-associated serine / threonine kinase 4 (Mast4) was one of the genes downregulated during chondrogenic differentiation of C3H10T1 / 2 mouse mesenchymal stromal cells and ATDC5 mouse chondrogenic cells ( Figure 1 a and Figure 8 To investigate the role of Mast4 in MSC differentiation, we used the CRISPR / Cas9 system to disrupt the Mast4 gene in C3H10T1 / 2 cells that undergo chondrogenic differentiation when BMP-2 or TGF-β1 is added ( Fig. 9 a, 9b) 22 We observed that in undifferentiated C3H10T1 / 2 cells, the expression of cartilage-specific genes was increased and the expression of matrix metallopeptidase (Mmp)-9 / 13 was decreased by CRISPR / Cas9-mediated Mast4 depletion and shRNA-mediated Mast4 knockdown ( Fig. 9 c, 9d). Furthermore, in high-density micromass cultures, the induction of cartilage matrix genes occurred earlier during differentiation into chondrocytes under BMP-2 stimulation and was enhanced in Mast4-depleted C3H10T1 / 2 cells ( Figure 1 b and Fig. 9 d). Interestingly, we noticed that the increase in Sox9 protein expression mediated by Mast4 depletion was more pronounced than Sox9 mRNA expression, implying a post-translational regulation of Sox9 by Mast4 ( Fig.10 ). Since Sox9 is a key transcription factor for chondrocyte-specific genes, the increase in Sox9 protein amount induced by Mast4 depletion may contribute to the maintenance of chondrogenic properties of C3H10T1 / 2 cells. Next, we generated Mast4-overexpressing C3H10T1 / 2 cells to examine whether Mast4 inhibits chondrogenesis. Due to the high molecular weight (>285 kDa) and relatively low expression of full-length Mast4, we instead used a truncated Mast4 construct (Mast4-PDZ) containing the DUF, kinase, and PDZ domains ( Fig.11a). We found that forced expression of Mast4-PDZ protein effectively inhibited the expression of chondrocyte marker genes ( Fig.11 b).

[0198] We further characterized the enhanced chondrogenesis induced by Mast4 depletion by RNA sequencing using wild-type and Mast4-depleted C3H10T1 / 2 micromass cultures treated with BMP-2 for 6 days. Differentially expressed gene (DEG) analysis identified 151 upregulated genes and 220 downregulated genes in Mast4-depleted C3H10T1 / 2 cells ( Fig.12 Gene ontology enrichment analysis and heat map analysis showed that genes related to chondrocyte differentiation (FDR = 1.01x10-3) and cartilage development (FDR = 5.05x10-6) were significantly enriched in Mast4-depleted cells ( Fig.12 and Figure 1 c). qRT-PCR confirmed the up-regulated genes in cartilage development ( Figure 1 d) and down-regulated genes in bone formation ( Figure 1 e) Expression in Mast4-depleted cells during chondrogenic and osteogenic differentiation. In addition, among collagen genes, cartilage-specific collagen genes (Col2a1, Col9a1, Col9a2, Col9a3, Col11a1, and Col11a2) were specifically upregulated upon Mast4 depletion ( Fig.13 In particular, in Mast4-depleted cells, 21 significantly upregulated genes related to cartilage and chondrocyte development and genes associated with BMP and TGF-β signaling pathways highly interacted, which play important roles in chondrogenesis, in addition to cartilage development in the transcriptional network ( Figure 1 f). Interestingly, Sox9 directly interacts with BMP2 and TGF-β1 and appears to function as a hub of a larger network as a node in both signaling pathways. Taken together, our data suggest that Mast4 can play a key role in regulating chondrogenic differentiation of MSCs.

[0199] Example 24.2 - Mast4 regulates chondrogenesis by modulating Sox9 stability

[0200] Next, we verified the effect of Mast4 on MSC chondrogenesis ( Fig.13 a). There was no significant difference in cell growth between wild-type cells and Mast4-depleted cells, whereas the cell growth of Mast4-PDZ-overexpressing C3H10T1 / 2 cells was significantly increased ( Fig.13 b). However, Alcian blue staining showed that chondrogenic differentiation was enhanced in Mast4-depleted cells, whereas overexpression of Mast4-PDZ inhibited chondrogenic differentiation ( Figure 2 a and Fig.13 c) 3D spheroid formation assay using low binding plates 23 It was also shown that spheroid size and expression of cartilage-specific genes increased upon Mast4 depletion ( Fig.13 d, 13e). In addition, chondrogenic differentiation of human bone marrow-derived stem cells (hBMSCs) depleted of MAST4 significantly increased the size of cartilage aggregates and increased the mRNA expression of COL2A1 and ACAN ( Figure 2 b and Fig.15 Chromatin immunoprecipitation (ChIP) analysis further revealed that during chondrogenic differentiation, Mast4 deficiency significantly increased the binding of Sox9 protein to the promoter region of the Col2a1 gene, while Mast4 overexpression reduced the binding of Sox9 protein to the promoter region of the Col2a1 gene ( Fig.16 a) It is noteworthy that even in the absence of Sox9 activation of Col2a1 gene transcription 18 In the presence of TGF-β1, Mast4 depletion was also sufficient to significantly increase the binding of Sox9 protein to the promoter region of the Col2a1 gene ( Figure 2 c). On the other hand, Mast4-PDZ overexpression reduced basal and TGF-β1-induced Col2a1 promoter activity and increased Sox9-induced Col2a1 promoter activity, which was more pronounced in Mast4-deficient cells ( Fig.16 b and Figure 2 d).

[0201] We observed that Mast4 depletion resulted in increased Sox9 protein expression ( Fig.10 ), which prompted us to investigate the post-translational regulation of Sox9 protein stability by Mast4. Given that Mast4 functions as a serine / threonine kinase, we examined whether Mast4 induces Sox9 protein phosphorylation, thereby leading to its protein degradation. We found that Mast4 binds to Sox9, and that overexpression of not only full-length Mast4 but also truncated Mast4 reduces Sox9 protein expression, whereas blocking Mast4 expression by siRNA restores Sox9 protein expression in a dose-dependent manner ( Figure 2 e, 2f and Fig.16 c). As expected, Sox9 serine phosphorylation was significantly reduced in Mast4-deficient cells, while basal Sox9 protein expression was increased, indicating that Mast4 can promote Sox9 degradation by inducing Sox9 phosphorylation ( Figure 2 g and Fig.16 d). Indeed, Mast4-induced Sox9 degradation was restored by MG-132 treatment ( Fig.16e). To identify the Mast4-mediated phosphorylation sites in Sox9 protein, we transiently overexpressed Sox9 and Mast4-PDZ proteins in C3H10T1 / 2 cells in the presence of MG-132 to prevent Mast4-mediated Sox9 degradation. Sox9 was then immunoprecipitated and then subjected to MASS SPEC analysis. MASS SPEC analysis showed that the phosphorylation status of serine 494 is regulated by Mast4 ( Figure 2 h). In particular, we observed that Mast4-induced Sox9 degradation was more pronounced in C3H10T1 / 2 cells treated with Sox9WT and the S494D substitution mutant of the mimic phosphorylated Sox9 protein, whereas the S494A substitution mutant of the Sox9 protein was hardly degraded by Mast4 ( Figure 2 i). We further found that the luciferase activity of the Col2a1 promoter reporter gene induced by Sox9 in wild-type cells was increased by Sox9S494A, which was comparable to the luciferase activity of Sox9WT in Mast4-depleted cells ( Figure 2 j). Interestingly, in the absence of Mast4, there was no significant difference in Col2a1 promoter activity between Sox9WT, S494A, and S494D. In addition, in cells transfected with Sox9S494A, the Mast4-PDZ-mediated reduction in Col2a1 activity was not significant ( Fig.17 We further observed that chondrogenesis was increased in C3H10T1 / 2 cells stably overexpressing Sox9S494A and decreased in Sox9S494D overexpressing cells by alcian blue dye staining and RT-PCR of chondrocyte marker genes ( Figure 2 k, 2l). These results suggest that Mast4-mediated phosphorylation of Sox9 protein at serine 494 can play an important role in the chondrogenic differentiation of MSCs.

[0202] Example 24.3 - TGF-β-induced inhibition of Mast4 enhances Sox9-Smad3 association

[0203] We observed an interaction between Mast4 and Smad3 proteins and an increase in TGF-β1 / Smad3-induced transcriptional activity in Mast4-deficient cells ( Fig.18 In addition, previous reports have shown that Smad3 enhances Sox9-dependent transcriptional activation during chondrogenesis through interaction with Sox9. 18Therefore, we hypothesized that Mast4 could inhibit TGF-β1 / Smad3-induced Sox9 transcriptional activity by promoting Sox9 protein degradation, resulting in reduced Smad3-Sox9 complex formation. In fact, both Sox9 protein stability and Smad3-Sox9 association increased with Mast4 depletion ( Figure 3 a). In addition, we found that Mast4 depletion increased Smad3 occupancy at the Sox9 binding site of the Col2a1 gene ( Figure 3 b). Importantly, the binding of Smad3 to the Smad7 promoter and the expression levels of TGF-β1 cellular repression target genes (e.g., Cdkn1a, c-myc, and Smad7) were not affected by the expression status of Mast4, indicating that TGF-β1-induced chondrogenic differentiation can be dependent on the level of Mast4 controlling the stability of Sox9 protein ( Fig.19 ).

[0204] Considering the role of TGF-β1 signaling in chondrogenic differentiation 12 We investigated whether TGF-β1 induces chondrogenesis by regulating the expression of Mast4. Interestingly, TGF-β1 treatment significantly suppressed the mRNA and protein expression of Mast4 ( Figure 3 c). TGF-β1 treatment also inhibited the activity of Mast4 promoter, but TGF-β1 treatment significantly increased Smad3 occupancy at Mast4 promoter, which means that TGF-β1 / Smad3 signaling can negatively regulate Mast4 transcription ( Figure 3 d, 3e and Fig. 20 In particular, a gradual decrease in Mast4 protein and mRNA expression levels was observed during chondrogenic differentiation of C3H10T1 / 2 cells and in human primary chondrocytes treated with TGF-β1, respectively ( Figure 3 f, 3g). These correlated well with increased Sox9 protein expression and increased mRNA expression of chondrocyte marker genes, reinforcing our finding of enhanced chondrogenesis in Mast4-deficient cells. Furthermore, treatment of C3H10T1 / 2 cells with the TGF-β receptor kinase inhibitor Vactosertib inhibited TGF-β signaling, prevented Mast4 gene downregulation, and blocked the induction of chondrocyte marker genes, indicating that TGF-β1 inhibition of Mast4 expression is essential for chondrogenic differentiation of MSCs ( Fig.21 ). Taken together, our observations suggest that TGF-β1 / Smad3 signaling promotes chondrogenesis by inhibiting Mast4 gene expression, leading to the subsequent accumulation of Sox9 protein.

[0205] Example 24.4 - Wnt-induced Mast4 stabilization increases MSC osteogenesis

[0206] It is widely accepted that Wnt / β-catenin signaling plays a key role in skeletal development by controlling the lineage commitment and differentiation of mesenchymal stromal cells into osteoblasts 19 Our observation that genes associated with osteogenesis were downregulated by Mast4 depletion in C3H10T1 / 2 cells led us to investigate whether Mast4 mediates Wnt / β-catenin-induced osteogenesis. Indeed, Alizarin Red S staining demonstrated that osteogenic differentiation of C3H10T1 / 2 cells was enhanced by stable overexpression of Mast4-PDZ ( Figure 4 a and Fig. 22 a, 22b). Interestingly, in undifferentiated MC3T3-E1 preosteoblasts, Wnt3a stimulation increased the expression of Mast4 ( Figure 4 b). In addition, osteogenic differentiation of MC3T3-E1 cells resulted in increased Mast4 expression as well as increased Wnt signaling, which was manifested by elevated levels of active β-catenin and inactive GSK-3β ( Fig. 22 c). We further observed that stable overexpression of Mast4-PDZ significantly increased β-catenin expression and subsequent Runx2 expression during osteogenic differentiation of C3H10T1 / 2 cells ( Figure 4 c). These observations were supported by RNA-seq analysis showing significant downregulation of osteogenesis-related genes in Mast4-depleted C3H10T1 / 2 cells ( Figure 1 c).

[0207] Evidence suggests that Mast4 functions as a potent mediator of Wnt / β-catenin-induced osteogenic differentiation of progenitor cells, and we found that GSK-3β reduced Mast4 protein expression in a dose-dependent manner, and GSK-3β inhibitor treatment significantly increased Mast4 expression ( Figure 4 d). In addition, GSK-3β inhibition and GSK-3β depletion significantly delayed the degradation of Mast4 ( Fig. 22 d, 22e). Since GSK-3β-induced phosphorylation has been found to regulate the stability of many target proteins 24 Therefore, we next addressed whether GSK-3β interacts with Mast4 to induce its degradation by phosphorylating Mast4. We observed that GSK-3β binds to the kinase domain of Mast4 and induces Mast4 serine phosphorylation ( Fig.23 a, 23b and Figure 4 e) In addition, in a GSK3 phosphorylation-dependent manner 24Among the reported E3 ubiquitin ligases recruited to target proteins, Smurf1 exhibited an interaction with the Mast4 kinase domain, the strength of which was regulated by the status of GSK-3β ( Fig.23 c and Figure 4 f). We further observed that GSK-3β overexpression enhanced Smurf1-mediated Mast4 degradation, but this was blocked by GSK-3β depletion ( Figure 4 g). Considering that WW domain-containing E3 ligases (including Smurf1) recognize the consensus PY motif (PPxY) 25 We found that mutation of the PY-like motif in the Mast4 kinase domain (P628A / Y634A) prevented Smurf1-mediated Mast4 degradation, which was no longer affected by GSK-3β depletion ( Figure 4 h). To map the regions responsible for GSK-3β-mediated degradation of Mast4 and Smurf1, we generated a series of deletion mutants of Mast4-PDZ (aa1-aa1229). Figure 4 As shown in Fig. 1, in the presence of GSK-3β, the expression level of the Mast4 depletion mutant containing the region between aa620 and aa1229 of Mast4-PDZ was significantly lower than that of the Mast4 depletion mutant shorter than aa620, indicating that the region between aa620 and aa1229 may contain the expected GSK3 consensus phosphorylation site (SxxxS p Interestingly, amino acid deletion at residues S632-S636 reduced Smurf1-mediated polyubiquitination of Mast4, which is increased by GSK-3β, while enhancing the stability of Mast4 ( Figure 4 j). Finally, Mast4 overexpression increased β-catenin nuclear localization and Runx2 transcriptional activity, which was further enhanced by blocking GSK3β regulation of Mast4 by S632-S636 deletion ( Figure 4 k, 4l). In addition, Mast4-PDZ overexpression or GSK-3β depletion led to a significant increase in Runx2 transcriptional activity and subsequent osteogenic differentiation of C3H10T1 / 2 cells ( Fig.24 Taken together, our data suggest that Mast4, stabilized by Wnt-mediated GSK-3β inhibition, can function as an important mediator of Wnt / β-catenin-induced osteogenesis.

[0208] Example 24.5 - Mast4 - / - Bone abnormalities in mice

[0209] To investigate the role of Mast4 in MSC differentiation, we generated Mast4 knockout genes using a CRISPR / Cas9-mediated knockout system. - / - Mouse ( Fig.25 a-25d). Although Mast4 - / - The mice were viable, but they were smaller than their wild-type littermates ( Fig.25 e). Although we cannot exclude the possibility that the effects of Mast4 on other organs also contribute to the small size of mice, we focused on investigating whether disruption of the Mast4 gene affects MSC differentiation during endochondral ossification in long bones, since MSCs are present in many visceral organs of adult mice. + / + Mast4 expression was observed throughout the tibial growth plate of mice ( Fig.26 a). Interestingly, increased expression of Mast4 was observed in hypertrophic chondrocytes, whereas a smaller fraction was localized in proliferating chondrocytes in the tibia. This observation supports our findings that Mast4 depletion enhances chondrogenic differentiation of C3H10T1 / 2 cells and suggests a role for Mast4 in mediating the switch to bone growth during the terminal stages of chondrocyte differentiation. In fact, Mast4 was expressed at 1 day PN. - / - Stronger expression of Sox9 and Col2a1 proteins (two key markers of chondrocytes) was observed in the growth plates of mice ( Figure 5 a, 5b). Pentachrome staining also showed that Mast4 - / - The mast cell layer in the proximal epiphysis of mice was increased ( Figure 5 c and Fig.26 b, 26c). In addition, at 3 weeks of PN, in Mast4 - / - More yellowish pentachrome staining was detected in the growth plates of mice, indicating that Mast4 depletion could facilitate collagen synthesis in the growth plates ( Figure 5 d) Mast4 - / - Tibial growth plate thickness in mice at 1d and 3 weeks after PN induction + / + There was no significant difference between the mice, but a significant decrease was observed at 6 weeks of PN ( Fig.26 b, 26d). However, at PN 3 and 6 weeks, Mast4 - / - The ratio of the mast cell layer to the total thickness of the growth plate was significantly increased in mice ( Fig.26 e). This observation suggests that Mast4 - / - Excessive cartilage synthesis in the mast cell layer of the mouse growth plate leads to reduced growth plate proliferation, resulting in abnormal osteogenesis. - / - and Mmp13 - / -Major phenotypes observed in mice 26 , so Mast4 - / - The increase in mast cell layers in mice may be related to the downregulation of Mmp9 and Mmp13 observed in Mast4-depleted C3H10T1 / 2 cells ( Figure 1 d and Fig. 9 c).

[0210] On the other hand, Mast4 - / - μCT analysis of mice revealed an osteoporotic phenotype with significantly reduced metaphyseal trabecular bone, more porous and thinner cortical bone, and reduced bone volume and mineral density ( Figure 5 e and Fig. 27 a, 27b). In addition, electron probe microanalyzer (EPMA) was used to analyze the 6-week-old Mast4 - / - The mice showed lower levels of key mineral ions for bone development and health, such as magnesium (Mg), phosphate (P), and calcium (Ca) in their tibiae ( Fig. 27 c) In particular, we observed that, compared with Mast4 + / + Compared with mice, Mast4 measured by double calcein labeling - / - The mice showed significantly reduced bone formation and shorter limb length ( Figure 5 f and Fig. 27 d) Consistently, at PN day 1, in Mast4 - / - Reduced expression of Osterix (an osteoblast marker essential for bone formation) and Mmp13 (a marker of mature osteoblasts and a target of Osterix) was observed in the proximal tibia and distal femur of mice. Figure 5 g and Fig.28 ).

[0211] We used the expression of cell surface markers 27 Further from Mast4 + / + and Mast4 - / - Skeletal stem cells were isolated from mice, namely CD45 - TER119 - TIE2 - ITGAV + THY1 - 6C3 - CD105 - The purified population ( Fig.29 Functional evaluation of these stem cells by in vitro colony formation assay revealed enhanced chondrogenic differentiation (shown as stronger Alcian blue staining intensity) but suppressed osteogenic differentiation (shown as weaker Alizarin Red S staining intensity), Mast4 - / - Mouse skeletal stem cell capacity ( Figure 5 h and Fig.30 We also isolated bone marrow-derived stem cells (BMSCs) and induced chondrogenic and osteogenic differentiation. - / - Enhanced chondrogenesis but reduced osteogenic differentiation was observed in mouse BMSCs ( Fig.31 ). Overall, our data suggest that Mast4 regulates MSC differentiation into cartilage or bone fate in vivo.

[0212] Example 24.6 - Mast4 - / - Cartilage phenotype in mouse tibia

[0213] To better understand the role of Mast4 in MSC differentiation, we collected and pooled Mast4 from 1-day PN cells. - / - RNA obtained from the tibia bones and cartilage of mice was subjected to RNA sequencing together with RNA from wild-type mice (three mice per group). Differential expression (DE) analysis showed tissue-specific expression, among which Mast4 - / - Mouse bones had 175 upregulated (CL1) and 181 downregulated (CL2) genes, and cartilage had 108 upregulated (CL4) and 327 downregulated (CL5) genes ( Fig.32 a). Gene ontology (GO) enrichment analysis revealed a high correlation with skeletal system development ( Fig.32 D in b; represented by CL2, CL3 and CL4; P = 4.5 × 10 -8 ~9.3×10 -4 ). Notably, 17 of the 120 common differentially expressed genes (DEGs; CL3) were primarily identified as Sox9 target genes, which are cartilage-specific for skeletal system development and show a clear shift in expression between bone and cartilage ( Fig.32 b), thus indicating that the function is involved in MSC differentiation. Gene set enrichment analysis of DEGs also revealed that a large number of DEGs in cartilage were assigned to cartilage development (FDR = 0.001), while DEGs in bones were assigned to skeletal system development (FDR = 0.004) and Wnt signaling pathway (FDR = 0.002) ( Figure 6 a, 6b). In particular, most Sox9 target genes are expressed in Mast4 - / - The expression of Mast4 in the cartilage of mice was upregulated, but not in the bone. - / - A network of Sox9 and Runx2 target genes showing differential expression in cartilage and / or bone of mice. These Sox9 and Runx2 targets highly interact with genes related to skeletal system development, including cartilage and bone development, TGF-β signaling, BMP signaling, and Wnt signaling ( Figure 6 c). Genes associated with cartilage development highly interacted with Sox9 target genes (i.e., Tgfb1, Bmp7, and Bgn; DE in cartilage). However, genes associated with skeletal development and Wnt signaling primarily interacted with common target genes of Sox9 and Runx2 (i.e., Nog, Bmp2, and Sox9; DE in bone) or Runx2 targets (i.e., Smad6; DE in bone). Interestingly, the Sox9 target gene network showed regulation of collagen family and CL3 switch genes in both bone and cartilage. Overall, our results indicate that Mast4 is a key regulator of transcriptional networks associated with skeletal system development.

[0214] Next, qRT-PCR was performed to identify wild-type and Mast4 - / - The expression of selected cartilage matrix genes and Sox9 target genes in the transcriptional network was further examined in cartilage tissue isolated from the tibia of mice ( Fig.33 a, 33b). In particular, downregulation of collagenase genes (Mmp9 and Mmp13) supports our conclusion that Mast4 - / - Observation of increased mast cell layer in mice ( Figure 5 c). We also found that in Mast4 - / - In the bone tissue of mice, the mRNA expression of genes related to osteoblast differentiation was reduced ( Fig.33 c, 33d). Consistent with these observations, in Mast4 - / - Increased expression of Sox9 and Col2a1 and decreased expression of β-catenin, Runx2, and Mmp13 proteins were observed in the cartilage and bone tissues of mice, respectively. Figure 6 d, 6e). In summary, we have - / - The results of molecular phenotype studies of mice support that Mast4 regulates the expression of the aforementioned genes by regulating Sox9, thereby ultimately promoting chondrogenesis in vivo.

[0215] Example 24.7 - Mast4 depletion promotes cartilage formation and repair

[0216] To further investigate the effect of Mast4 depletion on chondrogenesis in vivo, differentiated wild-type and Mast4-depleted C3H10T1 / 2 micromass cultures were implanted subcutaneously into nude mice to assess chondrogenesis ( Fig.34 a). After 2 weeks, transplanted tissues formed by Mast4-depleted cells showed a significant increase in volume ( Figure 7 a) Previous reports have shown that chondrogenesis of MSC implantation was barely observed via subcutaneous implantation, whereas it was more common via the use of diffusion chambers. 28 Or implanted into cartilage defect 29Similar to these reports, grafts formed by wild-type and Mast4-depleted C3H10T1 / 2 cells did not contain a high proportion of cartilage nodules. However, we found an increased incidence of cartilage nodule formation in grafts formed by Mast4-depleted cells. To confirm whether the nodules were composed of cartilage, tissue sections were stained with pentachrome ( Fig.34 b). In the grafts formed by Mast4-depleted cells, increased collagen deposition around the nodules was shown. In addition, Col2a1 (chondrogenesis marker protein) and Sox9 protein were highly expressed in the grafts formed by Mast4-depleted cells ( Figure 7 b). Taken together, these results indicate that depletion of Mast4 can enhance the chondrogenic commitment of MSCs in vivo.

[0217] Next, we evaluated the effect of MAST4 depletion in human bone marrow-derived stem cells (hBMSCs) on cartilage repair in a rabbit full-thickness cartilage defect model. Since hBMSCs are unable to form colonies from single cells, cell pools using CRISPR / Cas9-mediated MAST4 depletion showed at least 70% indels, frameshifts, or 21+bp indels. No abnormal findings or severe inflammatory responses were observed. While the defects in knee joints treated with vehicle (PBS) or transplanted with original hBMSCs were empty and distinct from the surrounding tissue, knee joints transplanted with MAST4-depleted hBMSCs exhibited smooth white repair tissue that covered the defect without showing a distinct edge with the normal surrounding cartilage ( Figure 7 c and Fig.35 ). The repair tissue structure of the MAST4-depleted hBMSC-transplanted knee joints was similar to that of the normal surrounding cartilage, while the repair tissue of the original hBMSC-transplanted knee joints exhibited irregular surfaces and obvious gaps in the boundary areas. In addition, increased safranin O and Masson's trichrome staining were noted in the repair tissue of the MAST4-depleted hBMSC-transplanted knee joints without obvious gaps, indicating increased production of cartilage tissue and collagen matrix, respectively. Immunohistochemical staining for type II collagen further revealed significantly strong expression and similar density by transplanted MAST4-depleted hBMSCs with surrounding normal tissue. Overall, the modified Wakitani score analysis 30 showed that transplantation of MAST4-depleted hBMSCs into defect sites significantly improved cartilage repair and regeneration ( Figure 7 d and Fig.36 ). Overall, these results suggest that Mast4 depletion may be a useful strategy for MSC-based therapy for cartilage regeneration. References

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[0282] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed within the scope of the appended claims.

Claims

1. A method for generating bone, comprising administering eukaryotic cells to a subject in need thereof at or near a bone defect site where bone formation is desired, wherein the expression or activity of microtubule-associated serine / threonine kinase family member 4 (MAST4) protein or a fragment thereof in the eukaryotic cells is stabilized or increased relative to normal cells.

2. The method according to claim 1, comprising recombinantly expressing MAST4 in a cell.

3. The method according to claim 1, wherein the cells are connective tissue cells.

4. The method of claim 3, wherein the cell is a mesenchymal stem cell, a fibroblast, an osteogenic progenitor cell, an osteocyte, a preosteoblast, an osteoblast, or an osteoclast.

5. The method of claim 1, wherein the cell is allogeneic with respect to the host. The method according to claim 2 , comprising recombinantly overexpressing MAST4 in a cell.

7. The method of claim 2, wherein the recombinantly expressed MAST4 is controlled by a viral promoter.

8. The method according to claim 7, wherein the viral promoter can be from a lentivirus or an adeno-associated virus.

9. The method of claim 1, wherein the cell is contacted with a composition comprising: (1) a compound that specifically binds to a nucleic acid encoding a MAST4 inhibitory protein, thereby inhibiting the expression of the MAST4 inhibitory protein; or (2) a compound that specifically binds to a MAST4 inhibitory protein, thereby preventing it from binding to MAST4.

10. The method according to claim 9, wherein the MAST4 inhibitory protein is of the GSK-3 family. The method of claim 10 , wherein the compound is a chemical substance, a polypeptide or a polynucleotide, or a combination thereof.

12. The method of claim 11, wherein the polypeptide is an antibody or an antigen binding molecule.

13. The method of claim 11, wherein the compound is a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi interacting RNA (piRNA), a small nuclear RNA (snRNA), or an antisense oligonucleotide, or a combination thereof.

14. The method of claim 11, wherein the compound is a CRISPR-Cas comprising a guide RNA specific for a nucleic acid encoding a MAST4 inhibitory protein (or a fragment thereof).

15. The method according to claim 14, wherein the guide RNA is a double RNA or a single-stranded guide RNA, the double RNA comprises CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), the double RNA is specific to a nucleic acid encoding a MAST4 inhibitory protein (or a fragment thereof), and the single-stranded guide RNA comprises a portion of crRNA and tracrRNA and hybridizes with a nucleic acid encoding a MAST4 inhibitory protein (or a fragment thereof).

16. The method according to claim 1, wherein the cells are connective tissue cells.

17. The method of claim 16, wherein the cell is a mesenchymal stem cell, a fibroblast, an osteogenic progenitor cell, an osteocyte, a preosteoblast, an osteoblast, or an osteoclast.

18. The method of claim 9, wherein the cell is allogeneic with respect to the host.

19. The method of claim 9, wherein the cell further comprises a recombinant construct expressing MAST4.

20. The method of claim 19, wherein the recombinant construct overexpresses MAST4.

21. A method for producing an extracellular matrix from eukaryotic cells, comprising contacting the eukaryotic cells with a composition comprising a compound that can specifically bind to a nucleic acid encoding a microtubule-associated serine / threonine kinase family member 4 (MAST4) protein or a fragment thereof and inhibit the expression or activity of the MAST4 protein, wherein the compound can specifically bind to a nucleic acid encoding the MAST4 protein or a fragment thereof, wherein the eukaryotic cells are chondrocytes, fibroblasts or mesenchymal stem cells.

22. The method of claim 21, wherein the compound is a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi interacting RNA (piRNA), a small nuclear RNA (snRNA), or an antisense oligonucleotide, or a combination thereof.

23. A method for preventing, treating or improving joint diseases, the method comprising (i) administering a compound to inhibit microtubule-associated serine / threonine kinase family member 4 (MAST4) in a eukaryotic cell, thereby inhibiting the expression or activity of the MAST4 protein; and (ii) administering the eukaryotic cells thus obtained to a subject in need thereof at or near a joint where cartilage formation is desired.

Images