Methods for treating osteoarthritis

Abstract

To provide methods for treating osteoarthritis by administering αKlotho protein and sTGFβ-R2 protein to a site within a mammal exhibiting symptoms of osteoarthritis, such as a knee joint.SOLUTION: A method of treating osteoarthritis in a subject in need thereof comprises administering to the subject a first viral vector comprising: a first nucleic acid sequence encoding an sTGFβ-R2 protein or an active fragment thereof; and a second nucleic acid sequence encoding an αKlotho protein or an active fragment thereof, thereby treating osteoarthritis in the subject.SELECTED DRAWING: Figure 1A

Description

[Background technology]

[0001] Articular cartilage is a tissue that undergoes substantial changes in matrix structure, molecular composition, metabolic activity, and mechanical properties during aging (see Rahmati M, Nalesso G, Mobasheri A, Mozafari M. Aging and osteoarthritis: Central role of the extracellular matrix. Ageing Research Reviews. 2017 Nov 1;40:20-30; Loeser RF, Collins JA, Diekman BO. Aging and the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 2016 Jul;12(7):412-20). As a result, articular cartilage experiences a decline in homeostasis and limited repair capacity, contributing to the development of osteoarthritis (OA) (Loeser RF, Collins JA, Diekman BO. Aging and the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 2016 Jul;12(7):412-20). Osteoarthritis is the most common musculoskeletal disorder among older adults and the leading cause of disability in the United States due to the pain associated with the disease (Zhang Y, Jordan JM. Epidemiology of Osteoarthritis. Clin Geriatr Med. 2010 Aug;26(3):355-69). Although symptomatic pain relief is possible for this disease (Zhang W, Ouyang H, Dass CR, Xu J. Current research on pharmacologic and regenerative therapies for osteoarthritis. Bone Research. 2016 Mar 1;4:15040), there is currently no available treatment to cure this condition.

[0002] The lack of effective clinical treatments for osteoarthritis supports the worldwide rise in the incidence of this condition (see Wittenauer R, Smith L, Aden K. Background Paper 6.12 Osteoarthritis. Background Paper. 2004;31). Currently, the most effective treatment for osteoarthritis other than arthroplasty is autologous chondrocyte transplantation. However, this treatment method has several limitations, including the need to harvest healthy donor cartilage through a separate surgical procedure, the limited proliferation capacity of primary chondrocytes, and the difficulty of treating large defects.

[0003] Therefore, there remains a need to find effective treatment methods that can avoid surgical procedures and treat this condition that is associated not only with aging but also with joint trauma. There is also a need to develop therapeutic targets to improve the low regenerative capacity observed in adults and that worsens with aging, and to help restore the structure and function of articular cartilage after osteoarthritis. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides a method for treating osteoarthritis in a mammal in need thereof by administering a therapeutically effective amount of an αKlotho protein or an active fragment thereof and a therapeutically effective amount of a sTGFβ-R2 protein or an active fragment thereof, or a combination thereof, to the mammal at a site exhibiting osteoarthritis in the mammal, wherein the progression of the osteoarthritis is inhibited compared to an untreated state, cartilage is increased, regenerated, or regrowth at the osteoarthritis site compared to an untreated state, or inflammation is inhibited compared to an untreated state. The osteoarthritis site is a site exhibiting symptoms of osteoarthritis. Osteoarthritis is the most common form of arthritis, affecting millions of people worldwide. Osteoarthritis occurs when the protective cartilage that cushions the ends of bones wears away over time. While osteoarthritis can damage any joint, the disorder most commonly affects joints in the hands, knees, hips, and spine. Symptoms of osteoarthritis include pain, stiffness, tenderness, loss of flexibility, grinding, bone spurs, and swelling.

[0005] A functional protein as described herein can be a full-length protein or a protein that differs from the full-length protein but retains, in whole or in part, the activity of the full-length protein.

[0006] Other features and advantages of particular embodiments of the invention will become more fully apparent from the following description of the embodiments and drawings thereof, and from the claims.

[0007] This patent or patent application file contains color drawings. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0008] [Figure 1A]Figure 1 shows the results of reproducing the early osteoarthritis phenotype in a rat model. A graph of the osteoarthritis grade of joints in rats based on the OARSI scoring system is shown (HC, n=5; OAC, n=5). Data are expressed as mean values, and each data point represents an individual rat. A two-tailed t-test (unpaired) was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 1B] Figure 1 shows the results of recapitulating the early osteoarthritis phenotype in a rat model. Representative Safranin O / Fast Green images of knee joints are shown (HC, n=5; OAC, n=5). The scale bar is 500 μm. These images show cartilage and matrix degradation 4 weeks after papain injection in the OAC group. [Figure 1C]

[0023] Figure 1 shows the results of recapitulating the early osteoarthritis phenotype in a rat model. Representative images of in situ cell death are shown (HC, n=3, OAC). Blue cells represent apoptotic cells. The scale bar is 20 μm. These images show that there are more blue cells in the OAC group. [Figure 1D] Figure 1 shows the results of recapitulating the early osteoarthritis phenotype in a rat model. Representative images of immunostaining detection of hypertrophy markers Col10a and RUNX2 in knee sections are shown (HC, n=3; OAC, n=3). Scale bar is 200 μm. Only the Col10 image contains DAPI co-staining (blue). The arrow in the Col10a image indicates the cartilage region characterized by the absence of Col10a in the non-diseased joint. [Figure 1E]

[0023] Figure 1 shows the results of recapitulating the early osteoarthritis phenotype in a rat model. Representative images of immunostaining detection of MMP13 in knee sections are shown (HC, n=3; OAC, n=3). The scale bar is 200 μm. The images show green staining in the nuclei for both groups, but only the OAC group shows staining outside the nuclear region. [Figure 1F]Figure 1 shows the results of recapitulating the early osteoarthritis phenotype in a rat model. Representative images of immunostaining detection of Sox9, Col2a, and ACAN in knee sections are shown (HC, n=3; OAC, n=3). The scale bar is 200 μm. Only the ACAN image contains DAPI co-staining (blue). [Figure 2A] Figure 1 shows the results of in vivo treatment with sTGFβR2 and αKlotho, which inhibits chondrocyte hypertrophy and promotes the upregulation of chondrocyte markers. Immunostaining quantification of Sox9, Col2a, and ACAN in tissue sections demonstrates the prevention of cartilage degradation and restoration of cartilage markers after treatment with sTGFβR2 and αKlotho as described herein. Quantification was performed within a 400 × 500 μm area along the cartilage region. Quantification was performed using Fiji software. HC = healthy (blue), OAC = diseased (red), Sham = untreated (green), and KT = treated (purple) (HC, n = 3; OAC, n = 3; KT, n = 3; and Sham, n = 3). A two-tailed t-test (unpaired) was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 2B]

[0023] Figure 1 shows the results of in vivo treatment with sTGFβR2 and αKlotho, which inhibits chondrocyte hypertrophy and promotes the upregulation of chondrocyte markers. Figure 2 shows an outline of the time course of osteoarthritis experiments. Female rats with papain-mediated osteoarthritis were sacrificed after 4 weeks (OAC) or injected intra-articularly with AAVDJ-GFP (Sham) or AAV-DJ-sTGFβR2 and AAV-DJ-αKlotho (KT). [Figure 2C]This figure shows the results of in vivo treatment with sTGFβR2 and αKlotho, which inhibited chondrocyte hypertrophy and promoted the upregulation of chondrocyte markers. Quantification of condylar cartilage thickness comparing four groups (HC, n=5; OAC, n=5; KT, n=5; and Sham, n=5) demonstrates how treatment with sTGFβR2 and αKlotho prevented hyaline cartilage destruction and promoted its formation. Condylar cartilage thickness was determined by measuring the thickness at three different locations in this cartilage region. Quantification was performed using Fiji software. A two-tailed t-test (unpaired) was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 3A] Figure 1 shows an experiment in which sTGFβR2 and αKlotho were delivered by the AAV-DJ serotype. Representative whole-body luminescence images (LUC, n=3; Sham, n=3) (left panel) and quantification of luminescence (arbitrary units, AU) 14 days after intra-articular injection of AAV-DJ-luciferase (LUC) or AAV-DJ-empty (Sham) are shown. [Figure 3B] Figure 1 shows an experiment in which sTGFβR2 and αKlotho were delivered by the AAV-DJ serotype. Flow cytometry analysis of rat synovial cells transduced in vitro with AAV-DJ-GFP showed a transduction efficiency of over 15% (top panel, untransduced cells; bottom panel, AAV-DJ-GFP-transduced cells) (n=3). [Figure 3C] Figure 1 shows an experiment in which sTGFβR2 and αKlotho were delivered by the AAV-DJ serotype. Flow cytometry analysis of rat chondrocytes transduced in vitro with AAV-DJ-GFP showed a transduction efficiency of less than 4% (left panel, untransduced cells; right panel, AAV-DJ-GFP transduced cells) (n=3). [Figure 4A]Figure 1 shows images demonstrating that intra-articular injection of sTGFβR2 and αKlotho promotes ECM repair and prevents apoptosis. Representative Safranin O / Fast Green images of knee joints showing higher cartilage and matrix degradation in the Sham group than in the OAC group (Sham, n=5; KT, n=5) (Scale bar, 500 μm). KT-treated knees show intact cartilage structure with high Safranin O staining. [Figure 4B] Images showing that intra-articular injection of sTGFβR2 and αKlotho promotes ECM repair and prevents apoptosis. Representative images of immunostaining detection of chondrocyte-specific markers Sox9, Col2a, and ACAN in knee sections are shown (Sham, n=3; KT, n=3). The scale bar is 200 μm. [Figure 4C] These images demonstrate that intra-articular injection of sTGFβR2 and αKlotho promotes ECM repair and prevents apoptosis. Representative images of in situ cell death are shown (Sham, n=4; KT, n=5). Blue cells represent apoptotic cells. The scale bar is 20 μm. These images show a higher number of blue cells in the Sham group. [Figure 4D] These images demonstrate that intra-articular injection of sTGFβR2 and αKlotho promotes ECM repair and prevents apoptosis. Representative images of immunostaining detection of hypertrophy markers Col10a and RUNX2 in knee sections are shown (Sham, n=3; KT, n=3). Scale bar is 200 μm. Only Col10 images contain DAPI co-staining (blue). [Figure 4E] These images demonstrate that intra-articular injection of sTGFβR2 and αKlotho promotes ECM repair and prevents apoptosis. Representative images of immunostaining detection of MMP13 in knee sections are shown (Sham, n=3; KT, n=3). The scale bar is 200 μm. The images show green staining in the cell nuclei for both groups, but only the Sham group shows staining outside the nuclear region. [Figure 4F]Figure 1 shows images demonstrating that intra-articular injection of sTGFβR2 and αKlotho promotes ECM repair and prevents apoptosis. Results of osteoarthritis grade of joints in rats based on the OARSI scoring system are shown (Sham, n=5; KT, n=5). Data are expressed as mean values, and each data point represents an individual rat. A two-tailed t-test (unpaired) was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 5A] This figure shows the results of sTGFβR2 and αKlotho suppressing osteoarthritis-associated immune responses in vivo. A bar plot of statistical enrichment scores of common DE genes (genes with common alterations in KT and HC but showing different behavior in other groups) between [KT vs. Sham] and [HC vs. OAC] by gene ontology enrichment analysis is shown. Only biological process terms with an FDR (false discovery rate) of less than 0.01 are shown in this plot. [Figure 5B] Figure 1 shows the results of sTGFβR2 and αKlotho suppressing osteoarthritis-associated immune responses in vivo. A bar plot of statistical enrichment scores of DE genes (genes not identified as DE in HC vs. OAC) in KT vs. Sham by gene ontology enrichment analysis is shown. Only biological process terms with an FDR (false discovery rate) of less than 0.01 are shown in this plot. FDR values ​​are shown on a -log10 scale. [Figure 5C] Figure 1 shows the results of sTGFβR2 and αKlotho suppressing osteoarthritis-associated immune responses in vivo. Gene expression heatmaps of downregulated DE genes that shared common changes in KT and HC but behaved differently in the other two groups are shown. Relative expression values ​​for each gene in various conditions were indicated by color (low = blue, high = red). Column dendrograms showed the results of hierarchical clustering based on the similarity of gene expression profiles. [Figure 5D]Figure 5 shows the results of sTGFβR2 and αKlotho suppressing osteoarthritis-associated immune responses in vivo. Gene expression plots of selected genes (from Figures 5C and 7) are shown. Gene expression was normalized to FPKM values ​​(Fragments Per Kilobase Per Million mapped reads), with average values ​​shown as bars and individual replicates shown as dots. The different conditions are represented by colors: HC (blue), OAC (red), Sham (green), and KT (purple) (HC, n=2; KT, n=2; OAC, n=3, and Sham, n=3). [Figure 5E] Figure 1 shows the results of sTGFβR2 and αKlotho suppressing osteoarthritis-associated immune responses in vivo. Gene expression plots for Nos2 (from Figure 7) are shown. Gene expression was normalized to FPKM values ​​(Fragments Per Kilobase Per Million mapped reads), with average values ​​shown as bars and individual replicates shown as dots. Different conditions are represented by colors: HC (blue), OAC (red), Sham (green), and KT (purple) (HC, n=2; KT, n=2; OAC, n=3, and Sham, n=3). [Figure 6A] Figure 1 shows the results of in vitro restoration of chondrocyte markers by sTGFβR2 and αKlotho. A schematic diagram of a co-culture assay (n=3) using human fibroblasts and human chondrocytes is shown. The scale bar is 200 μm. [Figure 6B] Figure 1 shows the results of in vitro recovery of chondrocyte markers by sTGFβR2 and αKlotho. Representative immunostaining images of Sox9 and Col2a derived from chondrocytes used in the co-culture experiment are shown (Sham, n=3; KT, n=3). The scale bar is 200 μm. [Figure 6C]This figure shows the results of in vitro restoration of chondrocyte markers by sTGFβR2 and αKlotho. The transduction of mesenchymal cells with AAV-DJ-sTGFβR2 and AAV-DJ-αKlotho improved chondrocytes, as demonstrated by immunostaining and quantification of Sox9, Col2a, and Ki67. Quantification was performed using Fiji software (Sham, n=3; KT, n=3). [Figure 6D] Figure 1 shows the results of in vitro restoration of chondrocyte markers by sTGFβR2 and αKlotho. Immunostaining quantification of Sox9, Col2a, and EdU demonstrated improved chondrocyte homeostasis in chondrocytes treated with sTGFβR2 recombinant protein and αKlotho recombinant protein. Quantification was performed using Fiji software (BSA, n=3; KT, n=3). A two-tailed t-test (unpaired) was used for statistical analysis in (b) and (c). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 7] Figure 1 shows heatmaps demonstrating that sTGFβR2 and αKlotho suppress the activation of immune response mechanisms associated with osteoarthritis. Heatmap of gene expression for DE genes that were differentially expressed between Sham and KT (but not between HC and OAC). Relative expression values ​​for each gene in various conditions are indicated by color (low = blue, high = red). Column dendrograms show the results of hierarchical clustering based on the similarity of gene expression profiles. [Figure 8] Figure 1 shows data on the grade (depth) of osteoarthritis in the knees of mice treated with the systemic gene therapy described herein. Group mean ± standard error of the mean (SEM). After two months of papain treatment, the control (Group 2) and STGFbR2 + FGF21 (Group 4) showed the highest grade, while the control (Group 1) showed the lowest grade after one month of papain treatment. A slight decrease in grade was observed in STGFbR2 + αKlotho (Group 3) compared with the control (Group 2) after two months of papain treatment. [Figure 9] Figure 1 shows data on the stage (range) of osteoarthritis in the knees of mice treated with the systemic gene therapy described herein. Group mean ± SEM. After two months of papain treatment, controls (Group 2) showed the highest mean OA stage, while STGFbR2 + FGF21 (Group 4) had a similar mean stage. After one month of papain treatment, controls (Group 1) showed a lower mean OA stage compared to controls (Group 2) after two months. STGFbR2 + αKlotho (Group 3) showed the lowest mean OA stage, demonstrating reduced lesion severity compared to the two control groups (Groups 1 and 2). [Figure 10] Figure 1 shows data on osteoarthritis scores (grade x stage) in the knees of mice treated with the systemic gene therapy described herein. Group mean ± SEM. The group treated with papain two months later (Group 2) and STGFbR2 + FGF21 (Group 4) had the highest scores, with no significant differences observed between these groups. The group treated with papain one month later (Group 1) had a lower score compared to the group treated with papain two months later (Group 2). STGFbR2 + αKlotho (Group 3) had the lowest score, indicating reduced lesion severity compared to the two control groups (Groups 1 and 2). [Figure 11] Figure 1 shows data on meniscal fibrosis in osteoarthritis of the knees of mice treated with the systemic gene therapy described herein. Group mean ± SEM. Meniscal fibrosis was observed in the two control groups (Groups 1 and 2) and STGFbR2 + FGF21 (Group 4), with Group 4 showing the most severe meniscal fibrosis score. STGFbR2 + αKlotho (Group 3) had no meniscal fibrosis. [Figure 12](A) Data showing gene expression of various genes in response to treatment with a combination of sTGFβR2 and αKlotho compared to treatment with sTGFβR2 individually and αKlotho individually. The combination synergistically achieved a higher therapeutic effect compared to the individual treatments. (B) Gel data demonstrating that chondrocytes treated with both factors showed higher protein expression of ACAN than chondrocytes treated with each factor separately. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure provides methods and compositions for treating or preventing osteoarthritis, such as osteoarthritis occurring in articular cartilage, using a therapeutically effective amount of a combination of αKlotho protein or an active fragment thereof and a soluble transforming growth factor beta receptor 2 (sTGFβR2) protein or an active fragment thereof, administered as a soluble protein to a mammal in need of such treatment or prevention, or a vector used to express the soluble protein at a site exhibiting osteoarthritis in the mammal. According to one embodiment, soluble transforming growth factor beta receptor 2 (sTGFβR2) is administered together with αKlotho protein to treat or prevent osteoarthritis in a joint, such as the knee, where articular cartilage is present. According to one embodiment, the soluble transforming growth factor beta receptor 2 (sTGFβR2) and αKlotho are administered separately or simultaneously so that both soluble transforming growth factor beta receptor 2 (sTGFβR2) and αKlotho are present at the administration site. According to one embodiment, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are encoded by multiple nucleic acids contained in one or more vectors, or are combined in a single viral vector, such as AAV, and are administered to treat or prevent osteoarthritis and / or diseases or symptoms associated with osteoarthritis.

[0010] According to one aspect, sTGFβR2 acts to inhibit TGFβ1, thereby suppressing osteophyte formation despite increased proteoglycan degradation (see Scharstuhl A, Glansbeek HL, van Beuningen HM, Vitters EL, van der Kraan PM, van den Berg WB. Inhibition of endogenous TGF-beta during experimental osteoarthritis prevents osteophyte formation and impairs cartilage repair. J Immunol. 2002 Jul 1;169(1):507-14). The TGFβ1 pathway controls cartilage homeostasis such that its balance and downstream effectors are essential for cartilage maintenance. On the other hand, TGF-β1 is thought to be essential for chondrogenesis due to its role in chondrocyte proliferation and maturation while avoiding chondrocyte hypertrophy (Yang X, Chen L, Xu X, Li C, Huang C, Deng CX. TGF-β / Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage. J Cell Biol. 2001 Apr 2;153(1):35-46).However, on the other hand, an increase in the ALK1 / ALK5 receptor ratio (see Blaney Davidson EN, Remst DFG, Vitters EL, van Beuningen HM, Blom AB, Goumans MJ, et al. Increase in ALK1 / ALK5 ratio as a cause for elevated MMP-13 expression in osteoarthritis in humans and mice. J Immunol. 2009 Jun 15;182(12):7937-45) or prolonged exposure to TGF-β1 significantly increases chondrocyte hypertrophy (see Pohlers D, Beyer A, Koczan D, Wilhelm T, Thiesen HJ, Kinne RW. Constitutive upregulation of the transforming growth factor-β pathway in rheumatoid arthritis synovial fibroblasts. Arthritis Research & Therapy. 2007 Jun 26;9(3):R59, Bakker AC, van de Loo FA, van Beuningen HM, Sime P, van Lent PL, van der Kraan PM, et al. Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthr Cartil. 2001 Feb;9(2):128-36).

[0011] According to this embodiment, soluble TGFβR2 (sTGFβR2), which lacks the membrane-binding domain and has high affinity for TGF-β1 and TGF-β3 (De Crescenzo G, Pham PL, Durocher Y, O'Connor-McCourt MD. Transforming Growth Factor-beta (TGF-β) Binding to the Extracellular Domain of the Type II TGF-β Receptor: Receptor Capture on a Biosensor Surface Using a New Coiled-coil Capture System Demonstrates that Avidity Contributes Significantly to High Affinity Binding. Journal of Molecular Biology. 2003 May;328(5):1173-83), may then modulate the effects of TGF-β1 in the joint.

[0012] According to one aspect, α-Klotho inhibits or prevents extracellular matrix (ECM) degradation (see Chuchana P, Mausset-Bonnefont AL, Mathieu M, Espinoza F, Teigell M, Toupet K, et al. Secreted α-Klotho maintains cartilage tissue homeostasis by repressing NOS2 and ZIP8-MMP13 catabolic axis. Aging (Albany NY). 2018 Jun 19;10(6):1442-53). Klotho was originally identified as an anti-aging gene in mice (see Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, et al. Suppression of aging in mice by the hormone Klotho. Science. 2005 Sep 16;309(5742):1829-33) and has been shown to be downregulated in cartilage and synovium during aging and osteoarthritis (see Pasztoi M, Nagy G, Geher P, Lakatos T, Toth K, Wellinger K, et al. Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts. Arthritis Research & Therapy. 2009;11(3):R68), and is classified as a type I membrane-bound protein with an extracellular domain that is released into the circulation by proteolytic cleavage (Xu Y, Sun Z. Molecular basis of Klotho: from gene to function in aging. Endocr Rev. 2015 Apr;36(2):174-93).The secreted protein αKlotho regulates surface glycoproteins such as ion channels, insulin-like growth factor 1 (IGF-1) / insulin, and Wnts by removing terminal sialic acids from N-linked glycans (see Dalton GD, Xie J, An SW, Huang CL. New Insights into the Mechanism of Action of Soluble Klotho. Front Endocrinol (Lausanne). 2017 Nov 17;8). αKlotho prevents apoptosis, oxidative stress, and immune responses in specific organs. (Fan J, Sun Z. The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells. Stem Cells. 2016 Jun;34(6):1615-25; Tilly EL, Vinatier C, Ong T, Guicheux J, Beck L. Role of the anti-aging protein Klotho in the autophagy- and senescence-associated development of osteoarthritis. Osteoarthritis and Cartilage. 2016 Apr 1;24:S64-5; Salech F, Varela-Nallar L, Arredondo SB, Bustamante DB, Andaur GA, Cisneros R, et al. Local Klotho enhances neuronal progenitor proliferation in the adult hippocampus. J Gerontol A Biol Sci Med Sci.) (2017 Dec 30).

[0013] According to one embodiment, there is provided a method for treating osteoarthritis in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a combination of αKlotho protein or an active fragment thereof and sTGFβ-R2 protein or an active fragment thereof at a site exhibiting osteoarthritis in the mammal, wherein the method results in inhibiting the progression of the osteoarthritis compared to an untreated state, increasing, regenerating, or regrowing cartilage at the osteoarthritis site compared to an untreated state, or inhibiting inflammation compared to an untreated state. According to one embodiment, the mammal is a dog or a human. According to one embodiment, the αKlotho protein or an active fragment thereof is administered as a soluble protein, and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein. According to one embodiment, the αKlotho protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection, and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection. According to one embodiment, a vector comprising a first nucleic acid sequence encoding αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding sTGFβ-R2 protein or an active fragment thereof is administered, and the first nucleic acid sequence is expressed to produce αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce sTGFβ-R2 protein or an active fragment thereof. According to one embodiment, a vector comprising a first nucleic acid sequence encoding αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding sTGFβ-R2 protein or an active fragment thereof is administered by intra-articular cartilage injection, and the first nucleic acid sequence is expressed to produce αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce sTGFβ-R2 protein or an active fragment thereof. According to one embodiment, a first vector containing a first nucleic acid sequence encoding αKlotho protein or an active fragment thereof and a second vector containing a second nucleic acid sequence encoding sTGFβ-R2 protein or an active fragment thereof are administered, and the first nucleic acid sequence is expressed to produce αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce sTGFβ-R2 protein or an active fragment thereof.According to one embodiment, a first vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second vector comprising a second nucleic acid sequence encoding a sTGFβ-R2 protein or an active fragment thereof are administered by intra-articular cartilage injection, and the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof. According to one embodiment, the vector is a recombinant virus. According to one embodiment, the vector is a parvovirus. According to one embodiment, the vector is an AAV vector. According to one embodiment, the AAV vector is AAV-DJ. According to one embodiment, the vector is an AAV vector classified as a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrh10.XX (where xx represents various variants known to those skilled in the art, such as AAVrh10.01 to AAVrh10.99, an example of which is AAVrh10.32), or a combination thereof. According to one embodiment, the vector infects mesenchymal cells at the site of osteoarthritis. According to one embodiment, the first vector and the second vector are recombinant viruses. According to one embodiment, the first vector and the second vector are parvoviruses. According to one embodiment, the first vector and the second vector are AAV vectors. According to one embodiment, the first vector and the second vector are AAV-DJ vectors. According to one embodiment, the first vector and the second vector are AAV vectors classified into the serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrh10.XX (where xx represents various known variants), or combinations thereof. According to one embodiment, the first vector and the second vector infect mesenchymal cells at the site of osteoarthritis. According to one embodiment, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins.According to one embodiment, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins. According to one embodiment, the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine proteins. According to one embodiment, the αKlotho protein has an amino acid sequence corresponding to SEQ ID NO: 1 below. [ka] The sequence has 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity, or 100% sequence identity to

[0014] According to one embodiment, the nucleic acid sequence encoding the αKlotho protein is the nucleic acid sequence encoding the αKlotho protein corresponding to SEQ ID NO: 2 below. [ka] [ka] [ka] The sequence has 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity, or 100% sequence identity to

[0015] According to one embodiment, the sTGFβ-R2 protein has the amino acid sequence of the sTGFβR2 receptor protein corresponding to SEQ ID NO: 3 below (the sTGFβR2 amino acid sequence is the bolded sequence in the IGG FC domain, and MGRGLLRGLWPLHIVLWTRIAST is the secretion signal): [ka] The sequence has 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity, or 100% sequence identity to

[0016] According to one embodiment, the nucleic acid sequence encoding the sTGFβ-R2 protein is the nucleic acid sequence encoding the sTGFβ-R2 protein corresponding to SEQ ID NO: 4 below. [ka] The sequence has 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more, or 100% sequence identity to

[0017] In one embodiment, the sTGFβ-R2 protein and / or the αKlotho protein is an Fc fusion protein comprising an Ig Fc domain, wherein the Ig Fc domain is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine Fc, or subtypes thereof, including IgG1, IgG2a, IgG2b, IgG3, and IgG4.

[0018] According to one embodiment, the Ig Fc domain has the amino acid sequence corresponding to SEQ ID NO: 5 below. [ka] a human Ig Fc domain having 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity, or 100% sequence identity to

[0019] According to one embodiment, the Ig Fc domain has the amino acid sequence corresponding to SEQ ID NO: 6: [ka] a mouse Ig Fc domain having 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity, or 100% sequence identity to

[0020] According to one embodiment, the Ig Fc domain has the amino acid sequence corresponding to SEQ ID NO: 7 below. [ka] a canine Ig Fc domain having 85% or more sequence identity, 86% or more sequence identity, 87% or more sequence identity, 88% or more sequence identity, 89% or more sequence identity, 90% or more sequence identity, 91% or more sequence identity, 92% or more sequence identity, 93% or more sequence identity, 94% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.5% or more sequence identity, or 100% sequence identity to

[0021] According to one embodiment, an amino acid sequence having a described homology to the αKlotho or sTGFβ-R2 protein amino acid sequence can be determined by obtaining a crystal structure of the αKlotho or sTGFβ-R2 protein, determining one or more active sites involved in binding or activity, and determining the homology of the structure that maintains useful binding or activity. Portions of the protein identified as inactive are suitable for amino acid substitution, modification, or mutation to create a protein with the claimed homology. Additionally, active portions may be modified, substituted, or mutated as long as useful binding or activity occurs. Ig Fc sequences having the desired homology can be determined in a similar manner. Methods for determining protein binding sites using X-ray crystallographic identification are known to those skilled in the art, including the general method described in Newcomer et al., PNAS, Vol. 90, pp. 9223-9227 (October 1993), and those skilled in the art can use or modify these methods to determine the binding site for αKlotho protein or sTGFβ-R2 protein. To determine 3D structures, binding pockets, tunnels and channels, surface features and cavities, and ligand-binding sites, the following software programs may be used: MED-SuMO (distributed by MEDIT), TRAPP (Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, Germany), CAVER (Masaryk University), GHECOM (open source), LIGSITEcsc, SURFNET, SiteHound, ICM-PocketFinder (Molsoft), SiteMap (Schrodinger), MSPocket (open source), POCASA (Hokkaido University), VOIDOO, FunFOLDQA (University of Reading), eFindSite (Louisiana State University), SiteEngine (Tel Aviv University), and SVILP_Ligand (Imperial College London).Useful databases include sc-PDB (University of Strasbourg), CASTp, Pocketome (an encyclopedia of conformational ensembles of druggable binding sites experimentally identifiable from co-crystal structures in the Protein Data Bank), PDBe motifs and sites, LigASite, PROtein SURFace Explore®, fPOP, PDBSITE (GeneNetworks), and LigBase (UCSF). Useful web services include 3DLigandSite (Imperial College London), metaPocket, PockDrug (University of Paris 7, France), PocketQuery (University of Pittsburgh), PASS, DEPTH, wwwPDBinder (University of Rome II, Italy), IsoMIF (University of Sherbrooke, Canada), LISE (Academia Sinica Institute of Biomedical Sciences), SiteHound-web (Sanchez Laboratory, Mount Sinai School of Medicine, New York), and MultiBind (Bioinformatics Group, Tel Aviv University).

[0022] An embodiment of the present disclosure provides a vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble transforming growth factor beta receptor II (sTGFβ-R2) protein or an active fragment thereof. According to one embodiment, a first promoter is operably linked to the first nucleic acid sequence for expression of the αKlotho protein or an active fragment thereof in a mammalian cell, and a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGFβ-R2 protein or an active fragment thereof in a mammalian cell. According to one embodiment, the first promoter and the second promoter are cell-specific or tissue-specific. According to one embodiment, the first promoter and the second promoter are constitutive or inducible.

[0023] The present disclosure provides a pharmaceutical formulation comprising, in a pharmaceutically acceptable excipient, a vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble transforming growth factor β receptor II (sTGFβ-R2) protein or an active fragment thereof.

[0024] The foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure.

[0025] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0026] In referring to the present disclosure, technical and scientific terms used in the description herein have the meanings that are commonly understood by those of ordinary skill in the art, unless specifically defined otherwise.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "protein" includes more than one protein, and reference to an "inactive ingredient" includes more than one inactive ingredient.

[0028] It should be further understood that the use of "or" means "and / or" unless otherwise indicated. Similarly, the terms "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. Also, where the term "comprising" is used in describing various embodiments, those skilled in the art will understand that in certain instances, the embodiments may instead be described using the words "consisting essentially of" or "consisting of."

[0029] As used herein, "gene" refers to a nucleic acid region that expresses a polynucleotide such as RNA, also called a transcribed region. A transcribed polynucleotide may have a sequence that encodes a polypeptide, such as a functional protein, which can be translated into the encoded polypeptide when placed under the control of appropriate regulatory regions. A gene may contain several segments, such as an operably linked promoter, 5' leader sequence, coding sequence, and 3' untranslated sequence, such as a polyadenylation site. A chimeric or recombinant gene is a gene not normally found in nature, such as a gene in which the promoter is not naturally associated with some or all of the transcribed DNA region. "Gene expression" refers to the process by which a gene is transcribed into RNA and / or translated into a functional protein.

[0030] "Gene delivery" or "gene transfer" refers to a method for the introduction of recombinant or foreign DNA into a host cell. The introduced DNA may remain unintegrated or, preferably, is integrated into the genome of the host cell. Gene delivery can occur, for example, by transduction using viral vectors or by transformation of cells using known methods such as electroporation, cell bombardment, etc.

[0031] "Transgene" refers to a gene that has been introduced into a host cell. A transgene may contain sequences that are native to the cell, sequences that do not naturally occur in the cell, or a combination thereof. A transgene may contain sequences that encode one or more proteins, which may be operably linked to appropriate control sequences for expression of the coding sequences in the cell.

[0032] "Transduction" refers to the delivery of a nucleic acid molecule into a recipient host cell by a gene delivery vector, such as, for example, an rAAV. For example, transduction of a target cell with an rAAV virion results in the introduction of the rAAV vector contained in the virion into the transduced cell. "Host cell" or "target cell" refers to the cell in which nucleic acid delivery occurs.

[0033] "Functional proteins" include variants, mutants, homologs, and functional fragments of the full-length protein. Based on the present disclosure, one of skill in the art would be readily able to construct proteins homologous to the full-length protein that retain the activity of the full-length protein in whole or in part.

[0034] A "vector" generally refers to a nucleic acid construct suitable for cloning and expressing a nucleotide sequence. One example of a vector is a viral vector. The term vector can also refer to a transport vehicle, such as a virus or virion, that contains a vector capable of introducing the vector into and between host cells.

[0035] "AAV vector" or "rAAV vector" refers to a recombinant vector derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAvDJ, or AAVrh10.XX. rAAV vectors may be deleted for one or preferably all wild-type AAV genes, but still contain functional ITR nucleic acid sequences. Functional ITR sequences are required for replication, rescue, and packaging of AAV virions. The ITR sequences may be wild-type sequences, or substantially identical sequences (defined below), or may be modified, for example, by nucleotide insertion, mutation, deletion, or substitution, so long as these sequences are functional.

[0036] A "therapeutically effective amount" refers to an amount effective, at the dosage and for the duration necessary, to achieve a desired therapeutic result, such as a result on osteoarthritis and related diseases or symptoms. A therapeutically effective amount of a parvovirus virion or pharmaceutical composition may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the parvovirus virion or pharmaceutical composition to induce a desired response in the individual. Dosage regimens may be adjusted to achieve an optimal therapeutic response. A therapeutically effective amount is also one in which any toxic or adverse effects of the parvovirus virion or pharmaceutical composition are typically outweighed by the beneficial effects of treatment.

[0037] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic outcome, such as prevention or inhibition of osteoarthritis. A prophylactic dose may be used in subjects prior to or at an early stage of disease, and in some cases, the prophylactically effective amount may be more or less than the therapeutically effective amount.

[0038] "Nucleic acid" includes any molecule composed of or containing monomeric nucleotides. The term "nucleotide sequence" may be used interchangeably with "nucleic acid" herein. A nucleic acid may be an oligonucleotide or a polynucleotide. A nucleic acid may be DNA or RNA. A nucleic acid may be a gene. A nucleic acid may be chemically modified or artificial. Artificial nucleic acids include peptide nucleic acids (PNAs), morpholino nucleic acids, and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Each of these is distinguished from natural DNA or RNA by modifications to the backbone of the molecule. Phosphorothioate nucleotides may also be used.

[0039] A "nucleic acid construct" is understood herein to mean an artificial nucleic acid molecule generated using recombinant DNA technology. A nucleic acid construct is a single- or double-stranded nucleic acid molecule that has been modified to contain nucleic acid segments combined and juxtaposed in a way that does not occur in nature. A nucleic acid construct is typically a "vector," i.e., a nucleic acid molecule used to deliver exogenously produced DNA into a host cell. One type of nucleic acid construct is an "expression cassette" or "expression vector." These terms refer to a nucleotide sequence that can cause the expression of a gene in a host cell or host organism that is compatible with such a nucleotide sequence. An expression cassette or expression vector typically contains at least appropriate transcription control sequences and optionally a 3' transcription termination signal. Additional factors necessary or beneficial for causing expression, such as expression enhancers, may be present. A nucleic acid construct may also be a vector that expresses or suppresses a protein by acting as RNA instead of DNA. To increase expression of a target protein, the nucleic acid construct may be an mRNA or analog that the cell, or more specifically, the ribosome, can recognize and generate multiple copies of the protein. When suppressing the expression of a target sequence, the RNA may be in a form that acts by preventing ribosomes from making the protein. This can be done by the mechanisms of RNAi, shRNA, miRNA, or Pri-miRNA. According to certain embodiments, suppression of a target sequence can be increased by delivering mRNA (or analogs) or shRNA (or analogs) that control the target sequence by suppressing known inhibitors of the target sequence. This can also be done by a vector that provides the DNA to be expressed, such as when using AAV.

[0040] "Operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are usually contiguous, and, where necessary to join two protein-coding regions, contiguous and in reading frame.

[0041] An "expression control sequence" refers to a nucleic acid sequence that controls the expression of a nucleotide sequence to which it is operably linked. An expression control sequence is "operably linked" to a nucleotide sequence if it regulates and controls the transcription and / or translation of that nucleotide sequence. Thus, expression control sequences can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, start codons before protein-coding genes, splicing signals for introns, 2A peptide sequences (allowing multicistronic expression), and stop codons. According to one embodiment, the first and second nucleic acid sequences encoding sTGFβ-R2 protein and αKlotho are separated by a polycistronic element. Polycistronic elements are generally understood to describe a type of messenger RNA that can separately encode two or more polypeptides within the same RNA molecule. The term "expression control sequence" is intended to include at least a sequence whose presence is designed to affect expression and may also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term may also include the design of a nucleic acid sequence to remove potential in-frame and out-of-frame undesirable initiation codons from the sequence. Expression control sequences may also include the design of a nucleic acid sequence to remove potential undesirable splice sites. Expression control sequences also include sequences that add a polyA tail (i.e., a chain of adenine residues at the 3' end of an mRNA), sometimes called a polyA sequence, i.e., a polyadenylation sequence (pA). Expression control sequences may also be designed to constitutively stabilize mRNA. Expression control sequences that affect transcriptional and translational stability, such as promoters, and sequences that confer translation suitable for use in insect cells, such as Kozak sequences, are well known to those skilled in the art. Expression control sequences may have properties that regulate the nucleotide sequence to which they are operably linked to achieve low or high expression levels.

[0042] Functional domains can also be fused to known proteins. This is the case when a mitochondrial signal is fused to CAT (catalase) so that acatalase is targeted for transport to mitochondria, where it performs its function inside or near the mitochondria instead of its natural location. Targeting signals can be added to other proteins to target them to other parts of the cell or to secrete them from the cell. For some proteins, the native sequence can be replaced with a more well-known version for improved efficacy, such as choosing the human or mouse secretion signal for TGFbR2 and fusing it to a canine version of the protein.

[0043] A "promoter" or "transcriptional control sequence" refers to a nucleic acid fragment that functions to regulate transcription of one or more coding sequences, is located upstream in the direction of transcription from a transcription start site of the coding sequence, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor protein binding sites, and activator protein binding sites, and any other nucleotide sequences known to those of skill in the art that act directly or indirectly to control the amount of transcription from the promoter (e.g., attenuators or enhancers, and silencers). A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by application of a chemical inducer. A "tissue-specific" promoter is active only in specific tissue or cell types. The present disclosure provides for operably linking a nucleic acid construct to a mammalian cell-compatible expression control sequence (e.g., a promoter). Many such promoters are known in the art (see Sambrook and Russell, 2001, supra). Constitutive promoters that are broadly expressed in many cell types, such as the CMV promoter and the hEf1α promoter, are disclosed. Variants of full-length hEf1α that are shorter than full-length hEf1α but still provide effective constitutive expression are also disclosed. Promoters that are inducible, tissue-specific, cell type-specific, or cell cycle-specific are disclosed. In disclosed embodiments, a nucleotide sequence encoding porphobilinogen deaminase is operably linked to a liver-specific promoter. Liver-specific promoters are particularly preferred for use with non-erythroid deaminases.Preferably, in the constructs of the present disclosure, the expression control sequence for liver-specific expression is selected from the group consisting of, for example, the alpha-1-antitrypsin (AAT) promoter, the thyroid hormone-binding globulin promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, the liver control region (HCR)-ApoCII hybrid promoter, the HCR-hAAT hybrid promoter, the AAT promoter combined with the mouse albumin gene enhancer (Ealb) element, and the apolipoprotein E promoter. Other examples include the E2F promoter for tumor-selective expression, particularly neural cell tumor-selective expression (Parr et al., (1997) Nat. Med. 3:1145-9), or the IL-2 promoter suitable for use in mononuclear blood cells (Hagenbaugh et al., (1997) J Exp Med, 185: 2101-10).

[0044] "3'UTR" or "3' untranslated sequence" (often also called the 3' untranslated region or 3' end) refers to nucleic acid sequences found downstream of the coding sequence of a gene, including, for example, transcription termination sites and (in most, but not all, eukaryotic mRNAs) polyadenylation signals (e.g., AAUAAA or variants thereof). After transcription is terminated, the mRNA transcript may be cleaved downstream of the polyadenylation signal, and a poly(A) tail may be added, which is involved in transport of the mRNA into the cytoplasm (where translation occurs).

[0045] As used herein, "native sequence" or "natural sequence" refers to a polynucleotide or amino acid that has been isolated from a natural source. A recombinant, naturally occurring polypeptide or naturally occurring polynucleotide having the same sequence as the native form is included in the "native sequence."

[0046] As used herein, "mutation" or "variant" refers to an amino acid sequence or polynucleotide sequence that has been altered by substitution, insertion, and / or deletion. In some embodiments, the mutated or variant sequence may have increased, decreased, or substantially similar activity or properties compared to the parent sequence.

[0047] "Percent sequence identity" and "homology" are used interchangeably herein to refer to comparisons between polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions where the identical nucleic acid base or amino acid residue is present in both sequences to count the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage sequence identity. Alternatively, the percentage may be calculated by determining the number of positions where the identical nucleic acid base or amino acid residue is present in both sequences, or the number of positions where the nucleic acid base or amino acid residue is aligned, including gaps to count the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage sequence identity. Those skilled in the art will appreciate that there are many established algorithms available to align two sequences.Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA), or by visual inspection (see generally Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)).

[0048] The BLAST and BLAST 2.0 algorithms are examples of suitable algorithms for determining percent sequence identity and sequence similarity, and are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analysis is publicly available on the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is terminated when the cumulative alignment score falls by the amount X from its maximum achieved value, when the cumulative score becomes 0 or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0049] The percentage degree of amino acid sequence identity can also be obtained by ClustalW analysis (vW1.8) by counting the number of perfect matches in the alignment, dividing the number of such perfect matches by the length of the reference sequence, and using the following default ClustalW parameters to achieve slow / accurate pairwise optimal alignment: Gap Open Penalty: 10, Gap Extension Penalty: 0.10, Protein weight matrix: Gonnet series, DNA weight matrix: IUB, Toggle Slow / Fast pairwise alignment = SLOW Alignment or FULL Alignment.

[0050] "Subject" or "patient" refers to a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey or human). The mammal may be a domestic animal, such as a dog, cat, mouse, cow, sheep, goat, horse, or pig. These mammals may be human subjects. In some embodiments, the human is an adult patient. In some embodiments, the human is a pediatric patient.

[0051] Delivery of nucleic acids encoding functional proteins Alternatively, foreign nucleic acid may be referred to as exogenous nucleic acid (i.e., nucleic acid that is not part of the cell's natural nucleic acid component) and may be introduced into a cell using any method known to those of skill in the art for such introduction. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, and conjugation. Those of skill in the art will readily understand and adapt readily identifiable references based on this disclosure. Foreign nucleic acid may be delivered to a subject by administering to the subject a nucleic acid or a vector comprising the nucleic acid described herein, for example, by systemic administration to the subject, such as by intravenous administration or injection, intra-articular administration or injection, intraperitoneal administration or injection, intramuscular administration or injection, intracranial administration or injection, intraocular administration or injection, or subcutaneous administration or injection.

[0052] Methods for gene therapy and delivery of genes to a subject, for example, using adeno-associated viruses, are described in U.S. Pat. No. 6,967,018, WO 2014 / 093622, U.S. Patent Application Publication No. 2008 / 0175845, U.S. Patent Application Publication No. 2014 / 0100265, EP 2432490, EP 2352823, EP 2384200, WO 2014 / 127198, WO 2005 / 122723, WO 2008 / 137490, WO 2013 / 14211, and the like. 4, WO 2006 / 128190, WO 2009 / 134681, EP 2341068, WO 2008 / 027084, WO 2009 / 054994, WO 2014059031, U.S. Pat. No. 7,977,049, and WO 2014 / 059029, each of which is incorporated by reference in its entirety, particularly as set forth in the respective patent or patent application, for methods describing delivery of genes to a subject.

[0053] vector Vectors are contemplated for use with the methods and constructs described herein. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors used to deliver nucleic acids to cells as described herein are known to those of skill in the art and include vectors used for such purposes. Specific exemplary vectors, among others, include plasmids, lentiviruses, and adeno-associated viruses, as known to those of skill in the art. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that contain one or more free ends, nucleic acid molecules that have no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retrovirus, lentivirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are inserted into the genome of a host cell upon introduction into that cell, and thereby are replicated along with the host genome. Certain vectors are also capable of expressing genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Common expression vectors utilized in recombinant DNA techniques are often in the form of plasmids.A recombinant expression vector can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, which can be selected based on the host cell used for expression. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0054] Non-viral delivery methods for nucleic acids or natural DNA-binding proteins, natural guide RNAs, or other natural species include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycationic or lipid-nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, which are incorporated herein by reference. Lipofection reagents are also available from commercial sources, such as Transfectam™ and Lipofectin™. Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in Felgner, WO 91 / 17424, and WO 91 / 16024. Delivery may be to a cell (e.g., in vitro or ex vivo administration) or to a target tissue (e.g., in vivo administration). The term native includes the protein, enzyme, or guide RNA species itself, but does not include the nucleic acid encoding those species.

[0055] In some embodiments, the gene therapy vector used in the methods herein is a parvovirus vector, e.g., an animal parvovirus, particularly a dependovirus, such as an infectious human or simian adeno-associated virus (AAV), and components thereof (e.g., an animal parvovirus genome) used as a vector for the introduction and / or expression of a nucleotide sequence encoding porphobilinogen deaminase in mammalian cells. Viruses of the Parvoviridae family are small DNA animal viruses. The Parvoviridae family can be divided into two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. Members of the Parvovirinae subfamily are referred to herein as parvoviruses and include the genus Dependovirus. As can be inferred from the genus name, members of the Dependovirus genus are unique in that they typically require co-infection with a helper virus, such as an adenovirus or herpesvirus, for productive infection in cell culture. The Dependovirus genus typically includes AAVs that infect humans (e.g., serotype 1, serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, and serotype 6) or primates (e.g., serotype 1 and serotype 4), as well as closely related viruses that infect other warm-blooded animals (e.g., bovine adeno-associated virus, canine adeno-associated virus, equine adeno-associated virus, and ovine adeno-associated virus). Additional information about parvoviruses and other members of the Parvoviridae family can be found in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996). For convenience, the present invention will be further illustrated and described herein with reference to AAVs. However, it should be understood that the present invention is not limited to AAVs and may be equally applicable to other parvoviruses.

[0056] The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank unique coding nucleotide sequences for nonstructural replication (Rep) and structural (VP) proteins. The VP proteins (VP1, VP2, and VP3) form the capsid. The terminal 145 nucleotides (nt) are self-complementary and organized to allow the formation of energetically stable intramolecular duplexes that form T-shaped hairpins. These hairpin structures serve as origins of viral DNA replication and as primers for the cellular DNA polymerase complex. Following wild-type (wt) AAV infection in mammalian cells, the Rep genes (i.e., Rep78 and Rep52) are expressed from the P5 and P19 promoters, respectively, and both Rep proteins function in viral genome replication. Splicing events within the Rep ORF actually result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, in mammalian cells, the unspliced ​​mRNAs encoding the Rep78 and Rep52 proteins have been shown to be sufficient for AAV vector production. Also, in insect cells, the Rep78 and Rep52 proteins are sufficient for AAV vector production.

[0057] As used herein, "recombinant parvovirus" or "AAV vector" or "rAAV vector" refers to a vector containing one or more polynucleotide sequences of interest, which are genes of interest or "transgenes," flanked by at least one parvovirus or AAV inverted terminal repeat (ITR) sequence. Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When rAAV vectors are incorporated into a larger nucleic acid construct (e.g., into a chromosome or into another vector, such as a plasmid or baculovirus, used for cloning or transfection), the rAAV vector, commonly referred to as a "provector," can be "rescued" by replication and encapsidation in the presence of AAV packaging and necessary helper functions. Thus, in another aspect, the present invention relates to a nucleic acid construct comprising a nucleotide sequence encoding a porphobilinogen deaminase as defined herein, wherein the nucleic acid construct is a recombinant parvovirus or AAV vector and therefore comprises at least one parvovirus or AAV ITR. Preferably, in the nucleic acid construct, the nucleotide sequence encoding the porphobilinogen deaminase is flanked on either side by the parvovirus or AAV ITRs.

[0058] AAV can infect many mammalian cells. See, e.g., Tratschin et al., (1985) Mol. Cell Biol. 5:3251-3260) and Grimm et al., (1999) Hum. Gene Ther. 10:2445-2450). However, transduction of human synovial fibroblasts by AAV is significantly more efficient than transduction of analogous murine cells (Jennings et al., (2001) Arthritis Res, 3:1), and the cellular tropism of AAV differs among serotypes. See, for example, Davidson et al. (2000) Proc. Natl. Acad. Sci. USA, 97:3428-3432, which discusses differences among AAV2, AAV4, and AAV5 in terms of mammalian CNS cell tropism and transduction efficiency, and Goncalves, (2005) Virol J. 2(1):43, which discusses approaches for modifying AAV tropism. In some embodiments, rAAV virions having AAV1 capsid protein, AAV8 capsid protein, and AAV5 capsid protein are preferred for transduction of liver cells (Nathwani et al., (2007) Blood 109(4):1414-1421; Kitajima et al., (2006) Atherosclerosis 186(1):65-73), of which rAAV virions having AAV5 capsid protein may be most preferred.

[0059] AAVs are highly prevalent in the human population (see Gao, G., et al., (2004) J Virol. 78(12):6381-8, and Boutin, S., et al., (2010) Hum Gene Ther. 21(6):704-12) and are useful as viral vectors. Many serotypes exist, each with tropism for different tissue types (see Zincarelli, C., et al., (2008) Mol Ther. 16(6):1073-80), allowing for preferential targeting of specific tissues by appropriate pseudotyping. Some serotypes, such as serotype 8, serotype 9, and serotype rh10, transduce the mammalian body. Zincarelli, C., et al., (2008) Mol Ther. 16(6):1073-80, Inagaki, K., et al., (2006) Mol Ther. 14(1):45-53, Keeler, AM, et al., (2012) Mol Ther. 20(6):1131-8, Gray, SJ et al., (2011) Mol Ther. 19(6):1058-69, Okada, H., et al., (2013) Mol Ther Nucleic Acids. 2:e95, and Foust, KD, et al., (2009) Nat Biotechnol. 27(1):59-65. AAV9 has been demonstrated to cross the blood-brain barrier, a difficult-to-reach site for many viral vectors and biologics (see Foust, KD, et al., (2009) Nat Biotechnol. 27(1):59-65, and Rahim, AA et al., (2011) FASEB J. 25(10):3505-18). Certain AAVs carry a 4.7-5.0 kb payload, including the viral inverted terminal repeats (ITRs) required in cis for viral packaging.See Wu, Z. et al., (2010) Mol Ther. 18(1):80-6, and Dong, JY et al., (1996) Hum Gene Ther. 7(17):2101-12, both of which are incorporated herein by reference.

[0060] AAV VP proteins are known to determine the cellular tropism of AAV virions. VP protein-encoding sequences are less conserved among different AAV serotypes than Rep proteins and genes. The ability of Rep and ITR sequences to cross-complement corresponding sequences in other serotypes allows for the production of pseudotyped rAAV particles containing capsid proteins from one serotype (e.g., AAV5) and Rep and / or ITR sequences from another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are part of the present invention. Herein, pseudotyped rAAV particles may be referred to as being of the "x / y" type, where "x" indicates the origin of the ITRs and "y" indicates the serotype of the capsid. For example, a 2 / 5 rAAV particle has ITRs from AAV2 and a capsid from AAV5. Modified "AAV" sequences can also be used in the context of the present disclosure, for example, for the production of rAAV vectors in insect cells. Such modified sequences include, for example, sequences having about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or more than about 70% nucleotide sequence identity and / or amino acid sequence identity to the ITR, Rep, or VP sequences of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAvDJ, or AAVrh10.XX (e.g., sequences having about 75% to about 99% nucleotide sequence identity), and can be used in place of the ITR, Rep, or VP sequences of wild-type AAV. Preferred adenoviral vectors are modified to suppress host responses. See, e.g., Russell (2000) J. Gen. Virol. 81:2573-2604, U.S. Patent Publication No. 20080008690, and Zaldumbide et al. (2008) Gene Therapy 15(4):239-46, all of which are incorporated herein by reference.

[0061] Regulatory Sequences and Terminators Regulatory sequences are contemplated for use with the gene therapy vector constructs described herein. The term "regulatory sequence" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control sequences (e.g., transcription termination signals such as polyadenylation signals and polyU sequences). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that constitutively express a nucleotide sequence in many types of host cells and those that express the nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired target tissue such as muscle, nerve, bone, skin, or blood, in a specific organ (e.g., liver or pancreas), or in a specific cell type (e.g., lymphocytes). Regulatory sequences can also direct expression in a time-dependent manner, e.g., cell cycle-dependent or developmental stage-dependent manner, and expression may be tissue- or cell type-specific. In some embodiments, vectors may contain one or more Pol III promoters (e.g., 1, 2, 3, 4, 5, or more Pol III promoters), one or more Pol II promoters (e.g., 1, 2, 3, 4, 5, or more Pol II promoters), one or more Pol I promoters (e.g., 1, 2, 3, 4, 5, or more Pol I promoters), or combinations thereof. Examples of Pol III promoters include, but are not limited to, the U6 promoter and the H1 promoter. Examples of Pol II promoters include, but are not limited to, the retroviral Rous Sarcoma Virus (RSV) LTR promoter (optionally with an RSV enhancer), and the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer).Examples of such enhancers include the SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter, as well as the Pol II promoters described herein. Enhancer elements such as the WPRE, CMV enhancer, the R-U5' segment in the LTR of HTLV-I (Takebe, Y. (1988) Mol. Cell. Biol. 8(1):466-472), the SV40 enhancer, and the intron sequence between exon 2 and exon 3 of rabbit β-globin (O'Hare K. et al., (1981) Proc. Natl. Acad. Sci. USA. 78(3):1527-31) are also encompassed by the term "regulatory sequence." One of skill in the art will understand that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the level of expression desired, etc. The vectors can be introduced into host cells to produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, variants thereof, fusion proteins thereof, etc.).

[0062] Embodiments of the methods described herein may utilize terminator sequences. Terminator sequences comprise nucleic acid sequence segments that mark the end of a gene or operon in genomic DNA during transcription. These sequences mediate transcription termination by providing a signal in newly synthesized mRNA that triggers processes that cause the mRNA to dissociate from the transcription complex. These processes involve direct interaction of mRNA secondary structures with the complex and / or indirect activity of recruited termination factors. Dissociation of the transcription complex frees RNA polymerase and associated transcription machinery to begin transcription of new mRNA. Terminator sequences are known in the art and include those identified and described herein.

[0063] Administration, Dosage, and Treatment In various embodiments, one or more gene delivery vectors, including viral vectors, and packaged viral particles containing the viral vectors may be in the form of a medicament or pharmaceutical composition and may be used to manufacture a medicament or pharmaceutical composition. The pharmaceutical composition may include a pharmaceutically acceptable carrier. Preferably, the carrier is suitable for parenteral administration. In certain embodiments, the carrier is suitable for intravenous, intraarticular, intraperitoneal, or intramuscular administration. Pharmaceutically acceptable carriers or excipients are described, for example, in *Remington: The Science and Practice of Pharmacy*, Alfonso R. Gennaro (Editor) Publishing Company (1997). Exemplary dosage forms may be in combination with sterile saline, glucose solution, or buffer, or other pharmaceutically acceptable sterile liquids. Alternatively, a solid carrier, such as microcarrier beads, may be used.

[0064] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for delivery of gene therapy vectors. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. The vectors of the present disclosure may be administered in sustained- or controlled-release formulations, for example, in compositions containing slow-release polymers or other carriers that will protect the compound against rapid release, including implants and microencapsulated delivery systems. For example, biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-polyglycolic copolymers (PLG).

[0065] In some embodiments, the gene therapy vector may be formulated with any acceptable carrier and administered parenterally, such as by intravenous administration, intraarticular administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, limb perfusion, or a combination thereof. Administration may be systemic, such that the gene delivery vector is delivered throughout the subject's body. In some embodiments, the gene delivery vector may be administered directly into the target tissue. In some embodiments, the gene delivery vector may be administered locally, such as by a catheter. The route of administration may be determined by one of skill in the art, taking into consideration, for example, the nature of the target tissue, the gene delivery vector, the intended therapeutic effect, and the maximum load that can be administered and absorbed by the target tissue.

[0066] Generally, an effective amount, particularly a therapeutically effective amount, of a gene delivery vector is administered to a subject in need thereof. A "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic outcome, such as treatment or amelioration of osteoarthritis, at the required dosage and for the required period of time. The effective amount or therapeutically effective amount of a vector may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the viral vector to elicit a desired response in the individual. The administration regimen may be adjusted to achieve an optimal therapeutic response.

[0067] In certain embodiments, the therapeutically or prophylactically effective amount of a nucleic acid, nucleic acid construct, parvoviral virion, or pharmaceutical composition ranges from 1×10 11 ~1×10 14 Genome copies (gc) / kg or 1 x 10 12 ~1×10 13 The dose may be expressed in genome copies (gc) / kg. Note that dosage values ​​may vary depending on the severity of the symptoms to be alleviated. Dosages may also vary based on the titer of the virion used. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the individual administering or supervising the administration of the composition. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by a physician.

[0068] The target tissue may be specific, for example, articular cartilage associated with osteoarthritis. In some embodiments, the effective dose range for small animals (mice) is 1×10 12 Genome copies (gc) / kg ~ 1 x 10 13 may be genome copies (gc) / kg, and 1 x 10 for large animals (cats or dogs) and human subjects 11 gc / kg~1×10 12 gc / kg, or 1 x 10 11 gc / kg~1×10 14 It may be genome copies (gc) / kg.

[0069] In various embodiments, the gene delivery vector can be administered as a bolus or by continuous infusion over a long period of time. In some embodiments, several divided doses can be administered over a long period of time, or the dose can be relatively reduced or increased depending on the urgency of the treatment situation. In some embodiments, the gene delivery vector can be administered daily, weekly, biweekly, or monthly. The treatment period can be one week or more, one month or more, two months or more, three months or more, six months or more, or eight months or more, or longer. In some embodiments, the treatment period can be up to one year or more, up to two years or more, up to three years or more, or indefinitely.

[0070] In some embodiments, a therapeutically effective amount of αKlotho protein or an active fragment thereof and a therapeutically effective amount of sTGFβ-R2 protein or an active fragment thereof are administered to a subject to treat osteoarthritis, e.g., osteoarthritis manifested by articular cartilage. The methods described herein treat or prevent disorders associated with osteoarthritis or symptoms of osteoarthritis, or improve the restoration of the structure and function of articular cartilage. The remission of osteoarthritis achieved by the administration of αKlotho protein or an active fragment thereof and sTGFβ-R2 protein or an active fragment thereof, directly or via the gene therapy methods described herein, is characterized by a suppression of symptoms in the subject compared to untreated subjects. In other aspects, gene therapy methods or the use of the nucleic acid vectors described above for use in the treatment or prevention of osteoarthritis in a subject are provided. According to one aspect, the administration of αKlotho and sTGFβR2 cooperatively suppresses or prevents the progression of osteoarthritis, e.g., by downregulating immune responses and promoting homeostasis and repair of joint tissue. [Example]

[0071] Example 1 Progression of histological changes associated with osteoarthritis in a rat model The present disclosure provides an animal model of osteoarthritis used in the experiments described herein. Osteoarthritis was mimicked by intra-articular injection of papain, a chemically induced model that disrupts cartilage microarchitecture and promotes proteoglycan degradation, affecting the integrity of the knee joint (see Pritzker KP. Animal models for osteoarthritis: processes, problems, and prospects. Ann Rheum Dis. 1994 Jun;53(6):406-20). This enzyme has no direct effect on collagen and chondrocytes, thereby not impairing mechanisms of cartilage regeneration that may be promoted by the tested treatments. This model reproduced several osteoarthritic phenotypes associated with osteoarthritis in animals and humans. For example, loss of ECM homeostasis caused by proteoglycan-degrading enzymes such as MMP13 is one of the major pathological features described in OA patients (see Troeberg L, Nagase H. Proteases involved in cartilage matrix degradation in osteoarthritis. Biochim Biophys Acta. 2011 Jul 8;1824(1):133-45).

[0072] Rat knee joints were analyzed 4 weeks after papain injection. Safranin O staining revealed clear signs of early osteoarthritis according to the standardized Osteoarthritis Research Society International (OARSI) score (see Pritzker KPH, Gay S, Jimenez SA, Östergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthr Cartil. 2006 Jan;14(1):13-29). Analysis revealed partial destruction of cartilage structure as well as the presence of MMP13 within the ECM as a result of papain treatment.

[0073] Rats (here, osteoarthritis control group, OAC) exhibited grade 2 osteoarthritis as defined by analytical parameters (see Figure 1A). Safranin O staining revealed reduced cartilage thickness with intermittent fibrous superficial and endochondral cell clusters (see Figure 1B). The osteoarthritis grade in these samples was further supported by TUNEL staining and immunohistochemistry, which demonstrated not only the presence of cell death (see Figure 1C) but also the presence of hypertrophic chondrocytes within the joint, according to elevated levels of Col10a and Runx2 (see Figure 1D), along with downregulation of the chondrocyte marker Sox9 (see Figure 1F and Figure 2A).

[0074] Col10a and Runx2 are well-known bone markers that, when found in chondrocytes, are associated with extracellular matrix (ECM) mineralization (see Chen D, Shen J, Zhao W, Wang T, Han L, L Hamilton J, et al. Osteoarthritis: toward a comprehensive understanding of pathological mechanism. Bone Research. 2017 Jan 17). Furthermore, the presence of proteolytic enzymes such as MMP13 within the matrix indicates cartilage damage and loss of joint function (see Xie Y, Mustafa A, Yerzhan A, Merzhakupova D, Yerlan P, N Orakov A, et al. Nuclear matrix metalloproteinases: functions resemble the evolution from the intracellular to the extracellular compartment. Cell Death Discov. 2017 Aug 14;3:17036). As a result, staining for aggrecan (ACAN) and type II collagen (Col2a) (see Figures 1F and 2A) showed a clear imbalance in the content of both matrix components within the cartilage compared to healthy knees that had not been treated with papain (healthy control group, HC). These results indicate that osteoarthritis developed in rats 4 weeks after papain treatment.

[0075] Example 2 Intra-articular injection using the AAV-DJ virus serotype To test the combined effects of αKlotho and sTGFβR2 on the progression and repair of osteoarthritis, these soluble factors were injected directly into the knee joint as a form of AAV vector-mediated gene therapy. It should be understood that each factor or its active fragment may be encoded by a separate nucleic acid and provided as a separate vector, or each factor may be encoded by a single nucleic acid and provided as a single vector. A single nucleic acid may be expressed to produce separate soluble factors, or a single nucleic acid may be expressed as a fusion protein of the soluble factors.

[0076] It should be understood that embodiments of the present disclosure contemplate the direct injection, delivery, or administration of the soluble factors αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof) to a patient in need of treatment. αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof) may be administered separately in multiple formulations, e.g., sequentially one after the other, or together, e.g., co-administered in the same formulation. It should be understood that embodiments of the present disclosure contemplate the direct injection, delivery, or administration of nucleic acids encoding the soluble factors αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof), e.g., nucleic acids in a vector such as AAV described herein for gene therapy, to a patient in need of treatment. The nucleic acid encoding αKlotho (or an active fragment thereof) and the nucleic acid encoding sTGFβR2 (or an active fragment thereof) may be administered separately in separate formulations, for example, sequentially one after the other, or may be administered together, for example, co-administered in the same formulation.

[0077] According to a particular embodiment, the dose of αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof) is 1×10 12 ~100×10 12 GC(AAV-DJ). An exemplary dose is 2.5 x 10 12GC(AAV-DJ) may be injected in 50 μl of PBS into the desired site, such as the knee, thereby providing a localized injection method.

[0078] We first performed intra-articular injection of AAV-DJ-luciferase to test the safety of this method. Luciferase measurements indicated that intra-articular injection limited AAV infection to the knee joint without invading the bloodstream, avoiding the effects on other tissues (see Figure 3A). This may help to avoid side effects. We first performed in vitro analysis using AAV-DJ-GFP to test the infectious efficacy of AAV-DJ serotypes. Flow cytometry analysis showed significantly higher transduction efficiency in synovial mesenchymal cells compared to chondrocytes, even though both populations were transduced (see Figures 3B and 3C). The high infection potency of mesenchymal stem cells may help to avoid deleterious cellular effects on chondrocytes as a result of direct AAV infection (Hermanns J, SCHULZE A, RR PJ-D, KLEINSCHMIDT JA, SCHMIDT R, HAUSEN HZ. Infection of Primary Cells by Adeno-Associated Virus Type 2 Results in a Modulation of Cell Cycle-Regulating Proteins. J Virol. 1997;71:8, Raj K, Ogston P, Beard P. Virus-mediated killing of cells that lack p53 activity. Nature. 2001 Aug;412(6850):914-7, Yang GS, Schmidt M, Yan Z, Lindbloom JD, Harding TC, Donahue BA, et al. Virus-Mediated Transduction of Murine Retina with Adeno-Associated Virus: Effects of Viral Capsid and Genome Size. J Virol. 2002 Aug;76 (15):7651-60). According to one embodiment, both αKlotho and sTGFβR2 are released by adjacent mesenchymal cells localized in the joint and exert their effects throughout the joint.

[0079] Example 3 αKlotho and sTGFβR2 improve clinical scores in osteoarthritic rats by preventing and reversing the osteoarthritic phenotype To test the possibility that αKlotho and sTGFβR2 are effective in cartilage repair, rats were treated with papain and allowed to develop early osteoarthritis for 4 weeks, after which they were treated by intra-articular injection of either AAV-DJ-GFP (Sham group) or AAV-DJ-αKlotho and AV-DJ-sTGFβR2 (KT group) (a schematic diagram can be seen in Figure 2B).

[0080] Rats injected with AAV-DJ-GFP showed greater cartilage deterioration after 6 weeks. Safranin O, Col2a, and ACAN staining revealed not only erosion and a clear loss of cartilage structure, as evidenced by the significant downregulation of ECM components in the remaining fragments (Figures 4A, 4B, and 2A), but also matrix mineralization. Immunohistochemical analysis revealed a significant decrease in the number of Sox9+ cells (Figures 4B and 2A). However, apoptotic cells (Figure 4C), hypertrophy markers (Figure 4D), and MMP13 (Figure 4E) were still present in the remaining cartilage segments. As a result, cartilage thickness was dramatically reduced (Figure 2C) (i.e., cartilage thinning as a symptom of osteoarthritis), and OARSI score analysis classified the injury as Grade 4 (Figure 4F), indicating clear progression to osteoarthritic pathology.

[0081] Rats treated with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2 showed significant phenotypic improvement after 6 weeks. Separate AAV-DJ viruses were generated using separate vectors. A total of 2.5 × 10 12GC was injected intra-articularly with 50% sTGFβR2 and 50% αKlotho. Intra-articular injection of AAV expressing αKlotho and sTGFβR2 not only prevented the release of MMP13 into the ECM but also promoted the maintenance of cartilage thickness. Compared to the OAC group, Safranin O staining (Figure 4A) demonstrated restoration of cartilage thickness (Figure 2C) and structure. First, the outermost layer, where cells were arranged in horizontal clusters parallel to the articular surface and organized into strings, pairs, and single cells. Second, the middle and deeper layers contained bilayers or multiple layers of chondrocytes arranged in vertical columns. Positive staining for Col2a and ACAN confirmed functional recovery of chondrocytes accompanied by regeneration of ECM components within the joint (Figures 1B and 2A). Suppression of ECM degradation by αKlotho was confirmed by the enhanced Col2a, ACAN, and Safranin O staining after KT treatment. This was further evaluated by analyzing histological sections. The data not only demonstrated the complete absence of apoptotic cells within the joints (see Figure 4C), but also demonstrated restoration of the distribution of hypertrophic markers. In contrast to the OAC and sham groups, Col10a- and Runx2-positive cells in KT-treated joints were mostly localized at the lower levels of the cartilage layer, corresponding to the regular hypertrophic layer of cartilage, similar to the HC group (see Figure 4D). Furthermore, the presence of the proteolytic enzyme MMP13 was absent within the ECM in HC- and KT-treated knees (see Figure 4E). Based on the significant joint improvement, the OARSI classification indicated that rats treated with αKlotho and sTGFβR2 recovered from grade 2 to grade 1 osteoarthritis, whereas rats treated with AAV-DJ-GFP further progressed to grade 4 osteoarthritis (see Figure 4F).

[0082] TGF-β / Smad signaling also contributes to the development and progression of osteoarthritis (see Shen J, Li S, Chen D. TGF-β signaling and the development of osteoarthritis. Bone Research. 2014 May 27;2:14002). Chondrocyte hypertrophy is promoted by an increase in the ALK1 / ALK5 receptor ratio during aging or long-term exposure to TGF-β1, demonstrating the importance of maintaining a balanced TGFβ pathway. Accordingly, the high affinity of the TGFβR2 receptor for TGFβ1 and TGFβ3 inhibits chondrocyte hypertrophy after KT treatment, resulting in the downregulation of hypertrophic markers.

[0083] In one embodiment, the use of both αKlotho and sTGFβR2 contributes to ECM restoration, for example, by balancing anabolic and catabolic pathways. The TGFβ1 pathway is believed to be a repair mediator by stimulating chondrocyte proliferation. In one embodiment, the use of sTGFβR2 to capture TGFβ1 suppresses the catabolic pathway but enhances its anabolic effects.

[0084] Example 4 .ALPHA.Klotho and sTGF.BETA.R2 ameliorate the inflammatory response characteristic of the osteoarthritis phenotype Although osteoarthritis was initially classified as a noninflammatory arthritis, it is characterized by synovial inflammation (see Scanzello CR, Goldring SR. The role of synovitis in osteoarthritis pathogenesis. Bone. 2012 Aug;51(2):249-57). Inflammation precedes the extensive loss of cartilage and joint space narrowing in osteoarthritic joints (Sokolove J, Lepus CM. Role of inflammation in the pathogenesis of osteoarthritis: latest findings and interpretations. Ther Adv Musculoskelet Dis. 2013 Apr;5(2):77-94).

[0085] To explore some of the mechanisms underlying the effects of αKlotho and sTGFβR2 on osteoarthritis, cartilage tissue was isolated from all groups for RNA-seq analysis. RNA-seq analysis revealed differentially expressed (DE) genes in the KT group compared with the OAC and sham groups. Specifically, 489 genes were differentially expressed in the KT group compared with the sham group, and 156 of these genes showed similar expression patterns between the KT and HC groups. Gene Ontology (GO) analysis indicated that among these differentially expressed genes, genes involved in inflammatory and immune responses showed the most dramatic effects upon KT treatment (see Figures 5A and 5B).

[0086] According to one aspect, chondrocytes are known to secrete pro-inflammatory cytokines in pathological conditions such as osteoarthritis (see Akkiraju H, Nohe A. Role of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration. J Dev Biol. 2015 Dec;3(4):177-92). Proinflammatory cytokines related to nuclear factor (NF)-κB and interleukin-1 (IL)-1β have been reported to promote the action of MMPs, which contribute to extracellular matrix degradation (see Raymond L, Eck S, Hays E, Tomek I, Kantor S, Vincenti M. RelA is required for IL-1β stimulation of matrix metalloproteinase-1 expression in chondrocytes. Osteoarthritis Cartilage. 2007 Apr;15(4):431-41; Liacini A, Sylvester J, Li WQ, Huang W, Dehnade F, Ahmad M, et al. Induction of matrix metalloproteinase-13 gene expression by TNF-α is mediated by MAP kinases, AP-1, and NF-κB transcription factors in articular chondrocytes. Exp Cell Res. 2003 Aug 1;288(1):208-17).Thus, when comparing the OAC and sham groups with KT, the data showed downregulation of (1) interleukin-related genes such as Il1rn (see Figure 5D and Figure 7), (2) Tnf-related / NF-κB-dependent genes such as Tnfaip2 (see Figure 5D and Figure 7), (3) interferon-related genes such as Ifit genes (see Figure 5C and Figure 5D), and (4) cytokines or chemokines such as Ccl6 (see Figure 5C and Figure 5D) (Appleton CTG, Pitelka V, Henry J, Beier F. Global analyses of gene expression in early experimental osteoarthritis. Arthritis Rheum. 2007 Jun;56(6):1854-68, Jeyakumar V, Halbwirth F, Niculescu-Morzsa E, Bauer C, Zwickl H, Kern D, et al. Chondrogenic Gene Expression Differences between Chondrocytes from Osteoarthritic and Non-OA Trauma Joints in a 3D Collagen Type I Hydrogel. Cartilage. 2017 Apr;8(2):191-8).

[0087] Thus, the data demonstrate that 4 weeks after papain treatment, chondrocytes already exhibit upregulation of pro-inflammatory cytokines and immune response-related factors. Treatment with αKlotho and sTGFβR2 not only downregulated the expression of some of these pre-expressed genes but also prevented the subsequent upregulation of other immune response factors, demonstrating a role for TGFβ in inflammation during osteoarthritis. TGFβ induces synovial lining cells to produce inflammatory factors, which can further stimulate hyaline chondrocyte hypertrophy (see Scanzello CR, Goldring SR. The role of synovitis in osteoarthritis pathogenesis. Bone. 2012 Aug;51(2):249-57). Blockade of TGF-β signaling significantly attenuated synovial thickening, which is involved in the pathogenesis of osteoarthritis (Scharstuhl A, Vitters EL, Kraan PM van der, Berg WB van den. Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor-β / bone morphogenetic protein inhibitors during experimental osteoarthritis. Arthritis & Rheumatism. 2003 Dec 1;48(12):3442-51). In addition, soluble Klotho regulates the PI3K / Akt pathway and the Wnt / β-catenin pathway, which are involved in cellular inflammatory responses.Various studies have shown how treatment with recombinant Klotho can reduce cytokine levels involved in kidney and heart disease (see Zhao Y, Banerjee S, Dey N, LeJeune WS, Sarkar PS, Brobey R, et al. Klotho Depletion Contributes to Increased Inflammation in Kidney of the db / db Mouse Model of Diabetes via RelA (Serine)536 Phosphorylation. Diabetes. 2011 Jul;60(7):1907-16; Hui H, Zhai Y, Ao L, Cleveland JC, Liu H, Fullerton DA, et al. Klotho suppresses the inflammatory responses and ameliorates cardiac dysfunction in aging endotoxemic mice. Oncotarget. 2017 Feb 1;8(9):15663-76). According to one embodiment, the cooperative activity of both αKlotho and sTGFβR2 reduced osteoarthritis-associated inflammatory responses.

[0088] During the inflammatory response that occurs during osteoarthritis, nitric oxide (NO) produced by Nos2 has destructive effects, causing chondrocyte death (see Vuolteenaho K, Moilanen T, Knowles R, Moilanen E. The role of nitric oxide in osteoarthritis. Scandinavian Journal of Rheumatology. 2009 Jul 12;Vol 36(4):247-58). NO, along with reactive oxygen species (ROS), appears to be a major inducer of chondrocyte death during osteoarthritis (see Del Carlo M, Loeser RF. Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species. Arthritis Rheum. 2002 Feb;46(2):394-403).

[0089] As a result, embodiments of the present disclosure relate to the use of αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof), either directly as soluble factors or via gene therapy, to prevent or reduce the subsequent destructive processes induced by a proinflammatory response. According to one embodiment (see Figures 5E and 7), KT treatment prevented the upregulation of this enzyme, which was significantly increased in sham animals at any time point after AAV injection. AAV-mediated expression of αKlotho and sTGFβR2 prevented cartilage degradation by reducing IL-1β-induced NO production through a reduction in Il1rn and Nos2 mRNA levels in chondrocytes. According to one aspect, αKlotho reduces oxidative stress and downregulates apoptosis upon KT treatment (Song S, Gao P, Xiao H, Xu Y, Si LY. Klotho Suppresses Cardiomyocyte Apoptosis in Mice with Stress-Induced Cardiac Injury via Downregulation of Endoplasmic Reticulum Stress. PLOS ONE. 2013 dic;8(12):e82968, Lin Y, Sun Z. Antiaging Gene Klotho Attenuates Pancreatic β-Cell Apoptosis in Type 1 Diabetes. Diabetes. 2015 Dec 1;64(12):4298-311, Maekawa Y, Ohishi M, Ikushima M, Yamamoto K, Yasuda O, Oguro R, et al. Klotho protein diminishes endothelial apoptosis and senescence via a mitogen-activated kinase pathway. See Geriatr Gerontol Int. 2011 Oct;11(4):510-6).

[0090] Example 5 .ALPHA.Klotho and sTGF.BETA.R2 promote the expression of human chondrocyte markers in vitro To assess the potential efficacy of KT treatment on human cartilage, we tested the in vitro effects of αKlotho and sTGFβR2 using human primary articular chondrocytes. The articular cartilage phenotype is characterized by the expression of cartilage-specific extracellular matrix components, primarily Col2a, and the cartilage-specific transcription factor Sox9. Sox9 expression is required for the commitment of mesenchymal cells to the chondrocyte lineage (see Lefebvre V, Dvir-Ginzberg M. SOX9 and the many facets of its regulation in the chondrocyte lineage. Connect Tissue Res. 2016 Apr 29;58(1):2-14). Maintenance of this differentiated phenotype in vitro is highly dependent on culture conditions. One of the major drawbacks associated with monolayer culture of these cells is the loss of the hyaline chondrocyte phenotype, which leads to chondrocyte dedifferentiation or hypertrophy (see Ma B, Leijten JCH, Wu L, Kip M, van Blitterswijk CA, Post JN, et al. Gene expression profiling of dedifferentiated human articular chondrocytes in monolayer culture. Osteoarthr Cartil. 2013 Apr;21(4):599-603).

[0091] Therefore, we used the two separate vectors described herein to test the effects of both αKlotho and sTGFβR2 on the phenotypic characteristics of human hyaline chondrocytes in monolayer culture. First, to mimic an in vivo model, we infected mesenchymal cells with viruses to bring them into close proximity with chondrocytes. To this end, we designed a coculture experiment as described herein in which human fibroblasts were efficiently infected with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2 (KT) or with AAV-DJ-GFP as a control (see Figure 6A). These results showed that KT-transduced mesenchymal cells exhibited a high percentage of chondrocytes expressing the chondrocyte-specific markers Sox9 and Col2a, which are essential for cell identity and ECM formation, respectively (see Figure 6B). The cultures also showed an increase in the number of cells undergoing cell cycling (see Figures 6B and 6C), confirming the effect of αKlotho on cell proliferation. Thus, a method is provided for re-growing cartilage by administering αKlotho or an active fragment thereof and sTGFβR2 or an active fragment thereof, or administering the genes in a vector for expression, for example, treatment with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2.

[0092] Human articular chondrocytes were also treated in vitro for 10 days with αKlotho and sTGFβR2. These results showed similar clear improvements, as evidenced by the induction of Sox9 and Col2a protein expression and enhanced cell proliferation (see Figure 6D). Thus, a method for treating human hyaline cartilage is provided by administering αKlotho or an active fragment thereof and sTGFβR2 or an active fragment thereof, or by administering the genes in a vector for expression, e.g., treatment with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2. Thus, αKlotho and sTGFβR2 are administered to maintain the cartilage phenotype in humans.

[0093] Example 6 Methods and Reagents Cell isolation and culture Human articular cartilage was collected from healthy donors with informed consent for use in clinical research. Rat articular cartilage was extracted from the femoral and tibial condyles of healthy rats under aseptic conditions. Chondrocyte isolation and culture were performed as previously described (see Gosset M, Berenbaum F, Thirion S, Jacques C. Primary culture and phenotyping of murine chondrocytes. Nat Protoc. 2008;3(8):1253-60).

[0094] Human fibroblasts (IMR90) were cultured in basal medium at 37°C and 5% CO2. Rat mesenchymal cells were isolated from the connective tissue of the joint capsule. Briefly, connective tissue was enzymatically digested (as described in (Yu G, Wu X, Kilroy G, Halvorsen Y-DC, Gimble JM, Floyd ZE. Isolation of murine adipose-derived stem cells. Methods Mol Biol. 2011;702:29-36)), and then stromal vascular cells were isolated by centrifugation and maintained in basal medium.

[0095] AAV cloning and production AAV plasmids were constructed following standard cloning techniques: α-Klotho and sTGFβR2 were PCR amplified using the following primers: [ka] [ka] [ka] The capital letters indicate the overlap with the secretion signal of the TGFβR2 receptor, and the bold letters indicate the AarI recognition site that creates the NotI overhang. [ka] The bold letters indicate the overlap with the extracellular domain of the TGFβR2 receptor, and the lowercase letters match the igg2a sequence used for overlap PCR. [ka] The bold letters indicate the overlapping portion with the mouse Igg2a FC region, and the lowercase letters match the sequence of the extracellular domain of TGFβR2 used in overlap PCR. [ka] In bold is the AarI recognition site that creates an NheI overhang.

[0096] AAV was prepared using 293AAV cells (Cell Biolabs, Inc.) as described with minor modifications (see Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno-associated viral vectors. Nat Protoc. 2006;1(3):1412-28). Briefly, cells were transfected using calcium phosphate, and virus was purified through a CsCl gradient. Viral titers were determined by qPCR using the following primers: [ka]

[0097] Osteoarthritis injury model Experimental osteoarthritis was induced by intra-articular injection of 100 μl of 4% papain (Sigma-Aldrich, P4762) prepared in PBS, followed by 50 μl of 0.03 M L-cysteine ​​(Sigma) prepared in PBS. Both solutions were filtered through a 0.22 μm filter before injection. These injections were performed three times (days 1, 4, and 7). All animal experiments were performed in accordance with protocols approved by the IACUC and the Salk Institute Animal Resources Department.

[0098] Animal experiment design Twenty 250 g female Sprague-Dawley (SD) rats were divided into four groups, each consisting of eight animals: healthy control (HS), osteoarthritis control (OAC), osteoarthritis treated with αKlotho and sTGFβR2 for 4 weeks (KT), and osteoarthritis sham (Sham) for 4 weeks. OAC, KT, and Sham rats were treated with papain / cysteine.

[0099] Four weeks after the last papain / cysteine ​​injection, the OAC rats were sacrificed to determine the osteoarthritis grade achieved at this time point. Two other groups received intra-articular AAV treatment: AAV-DJ-GFP was injected into the sham group, and AAV-DJ-αKlotho and AAV-DJ-sTGFβR2 were injected into the KT group. A total of 2.5 × 10 mice were injected in 50 μl of PBS per knee. 12 The final two groups were then injected with GC. These last two groups were sacrificed 6 weeks after virus injection. Both knee joints were collected from each rat: one knee for histological analysis and the other for RNA isolation. All animal experiments were performed in accordance with protocols approved by the IACUC and the Salk Institute Animal Resources Department.

[0100] RNA extraction The cartilage surface was washed with saline and then dissected from the articular surface using a razor blade. Care was taken to avoid contamination with blood, bone, or synovial membrane. The tissue was cut into small pieces, immersed in TRIzol (Ambion), and immediately flash-frozen and stored at -80°C until further use. Total RNA was isolated from the cartilage tissue using the TRIzol method. To determine the quality and integrity (RIN) of the total RNA samples, each RNA sample was run on a TapeStation automated electrophoresis analysis system (2200 TapeStation) according to the manufacturer's instructions. RNA concentration was determined using a Qubit Fluorometer 2.0.

[0101] Histology and immunofluorescence Whole knee joints were prepared for histology as described in Kawamoto and Shimizu, 2000 (see Kawamoto T, Shimizu M. A method for preparing 2- to 50-μm-thick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem Cell Biol. 2000 May 1;113(5):331-9). Samples were cut into 7-μm-thick slices using the method described in Kawamoto and Kawamoto, 2014 (see Kawamoto T, Kawamoto K. Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamoto's film method (2012). Methods Mol Biol. 2014;1130:149-64). Sections were then stained using various methods. Safranin O / Fast Green staining was performed according to standard techniques. The Osteoarthritis Research Society International (OARSI) scoring system was used to grade the pathological changes in articular cartilage according to previously described methodology (23): Grade 0 was assigned to normal and healthy cartilage; Grade 1 was applied when the cartilage surface was intact but contained areas of wear, hypertrophy, and cell clusters; Grade 1.5 included cell death; Grade 2 cartilage showed an intermittent fibrous surface; Grade 2.5 consisted of Grade 2, which included matrix loss as indicated by less than one-third of the cartilage staining with Safranin O; Grade 3 was determined when cracks appeared down to the middle layer and less than two-thirds of the cartilage was stained with Safranin O; Grade 3.5 indicated deeper cracks into the middle layer; Grade 4 indicated matrix loss with delamination of the outermost layer; Grade 4.5 indicated indentation into the middle layer; Grade 5 cartilage indicated completely eroded, non-calcified cartilage; Grade 5.5 indicated hypertrophic cartilage growth after erosion; and higher grades, Grade 6, indicated more severe cartilage damage when condylar deformation was present.

[0102] The thickness of the entire condylar cartilage was measured using the image analysis software ImageJ. Samples were evaluated by two blinded investigators by examining three different locations along the length of the cartilage.

[0103] For immunofluorescence analysis, sections were stained with antibodies and counterstained with 4,6-diamidino-2-phenylindole (DAPI), and images were captured using a slide scanning microscope (Olympus VS-120 Virtual Slide Scanning Microscope).

[0104] Apoptosis detection was performed on rat knee sections using the In situ cell death detection AP kit (Roche) according to the manufacturer's protocol.

[0105] antibody The antibodies used were type II collagen (NeoMarkers) at a 1:100 (v / v) dilution, Runx2 (Santa Cruz Biotechnology) at a 1:100 (v / v) dilution, MMP13 (Abcam) at a 1:100 (v / v) dilution, MMP3 (Abcam) at a 1:100 (v / v) dilution, Sox9 (Abcam) at a 1:100 (v / v) dilution, Collagen X (Abcam) at a 1:50 (v / v) dilution, and Ki67 (BioLegend) at a 1:100 (v / v) dilution. Heat-mediated antigen retrieval was performed with 5% hyaluronidase in acetate buffer at 37°C for 1 h. Immunoreactivity was visualized with a biotinylated anti-mouse IgG secondary antibody using an avidin / biotin blocking kit (Vector Laboratories) according to the manufacturer's protocol.

[0106] In vivo luciferase detection Six 300g Long-Evans rats were tested for intra-articular injection of AAV-DJ, i.e., AAV-DJ-Luc or AAV-DJ (empty vector, negative control). Luciferase was detected using an IVIS Kinetic 2200 (Caliper Life Sciences) two weeks after injection. 50mg / kg of D-luciferin (Biosynth) was injected intra-articularly and intraperitoneally. Images were acquired 10 minutes after D-luciferin injection.

[0107] RNA sequencing and data analysis Reads were mapped to reference rn6 (Illumina iGenomes) using STAR [v2.5.1b (Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013 Jan 1;29(1):15-21)] using default parameters. Only uniquely mapped reads were used for downstream analysis. Gene expression levels were calculated by summing mapped reads across all exons of RefSeq genes using HOMER [v4.8 (Homer Software and data download [internet]. Available on the World Wide Web at homer.ucsd.edu / homer)]. Differentially expressed (DE) genes were identified using DESeq2 [v1.18.1 (Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 [Internet], available on the World Wide Web at ncbi.nlm.nih.gov / pmc / articles / PMC4302049 / )] using a cutoff of logFC (log fold change) > 0.5 and FDR (false discovery rate) < 0.05.

[0108] Enrichment studies were performed using DAVID (v6.8) (see Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009 Jan;37(1):1-13), using the gene of interest as input signal and the entire rn6 genome as background. Only biological process terms with Benjamini-Hochberg FDR < 0.01 were used. Data can be accessed through GEO accession number GSE118559.

[0109] Unless specifically mentioned, all statistical analyses of RNA-seq were performed in the R environment [v3.4.3, (R: A Language and Environment for Statistical Computing [Internet]. Available on the World Wide Web at gbif.org / tool / 81287 / ra-language-and-environment-for-statistical-computing)]. Figures were plotted using the R packages ggplot2 (ggplot2 - Elegant Graphics for Data Analysis | Hadley Wickham | Springer [Internet]. Available on the World Wide Web at springer.com / us / book / 9780387981413) and gplots (see Wickham H, Chang W, Henry L, Pedersen TL, Takahashi K, Wilke C, et al. ggplot2: Create Elegant Data Visualizations Using the Grammar of Graphics [Internet]. Available on the World Wide Web at CRAN.R-project.org / package=ggplot2).

[0110] In vitro experiments Co-culture experiments: The trans-in vitro effects of sTGFβR2 and αKlotho on human chondrocytes were evaluated using Corning® Transwell® polyester membrane cell culture inserts (Sigma) with 4 μm pore size. Briefly, 3 × 10 human fibroblast cells were plated on p100 plates. 12 The cells were transduced with AAV-DJ-αKlotho and AAV-DJ-TGFβR2. Two days after transduction, the cells were replated in the upper well of the co-culture chamber, and on the same day, chondrocytes were seeded in the lower well of the chamber. The cells in the chamber were cultured at 37°C in 5% CO for 10 days.

[0111] Soluble factor experiments: Chondrocytes were plated at 60% cell density and treated with BSA or 5 ng / ml and 10 ng / ml of αKlotho (Abcam ab84072) and sTGFβR2 (R&D Systems 241R2025) for 10 days. Cells were cultured at 37°C in 5% CO2, and the medium containing the factors or BSA was changed every 3 days.

[0112] Immunofluorescence of cell cultures Before fixation, cells were incubated with EdU for 2 hours according to standard protocols. Then, cells were fixed using 4% PFA. Cells were permeabilized with 0.1% Triton X-100 in PBS for 20 minutes at room temperature (RT). After washing with PBS, cells were blocked with 1% BSA in PBS for 1 hour and then incubated with primary antibodies (type II collagen diluted 1:150 and Sox9 diluted 1:300) overnight at 4°C. Secondary antibodies were incubated for 1 hour at room temperature. EdU staining was performed using the Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit (Invitrogen). Counterstaining was performed using DAPI. Images were acquired using a Zeiss LSM 880 Rear Port Laser Scanning Confocal microscope.

[0113] Flow cytometry analysis After filtering (70 μm) and washing with 2% FBS / PBS, GFP-positive cells were detected using a FACS Canto II (BD Biosciences).

[0114] statistics Quantitative data are expressed as mean ± standard error (se). Statistical significance ( ) was determined by unpaired two-tailed Student's t test with Welch's correction (no assumption of equal sd for each group). * P values ​​were determined. All analyses were performed using Prism7 software from GraphPad (San Diego, CA, USA). Statistical significance was determined as P<0.05, P<0.05, P<0.05. ** P<0.01, *** P<0.001, **** P<0.0001 was determined.

[0115] Example 7 Gene therapy for osteoarthritis using systemic injection of sTGFbR2+αKlotho Osteoarthritis (OA) lesions induced by intra-articular injection of papain as described herein consisted primarily of an intact cartilage surface with superficial fibrosis, chondrocyte death / loss and proliferation, edema, and / or loss of the proteoglycan matrix in the most superficial layer. Occasionally, deeper fibrosis, wear, and fissures extending into the middle layer were observed. Superficial fibrosis was also observed along the surface of the meniscus. Fibrosis was characterized by small cracks and discontinuities in the most superficial layer or meniscus surface and discontinuities in the cartilage matrix. Chondrocyte death / loss was characterized by the absence of chondrocytes or "ghost" chondrocytes in the most superficial and middle layers of cartilage. Chondrocyte proliferation was characterized by an increased number of chondrocytes, often in a disorganized fashion, within the most superficial and middle layers of cartilage. Edema was characterized by an increase in clear fluid surrounding chondrocytes in the most superficial and middle layers of cartilage. Proteoglycan matrix loss was characterized by a decrease or loss of cationic stain (red, Safranin O). Matrix loss could occur immediately adjacent to viable chondrocytes or in areas of chondrocyte loss and was usually associated with other lesions as described above. Wear was characterized by focal loss of the outermost layer, resulting in a rough surface. Cracks were characterized by longitudinal matrix separation extending into the intermediate cartilage layer. To evaluate gene therapy for osteoarthritis in rat knees, rats were systemically injected with viral vectors containing nucleic acids encoding sTGFbR2 and FGF21.

[0116] As shown in Figure 8, OA grade was determined based on the depth of the most severe lesion found in the specimen (superficial, intermediate, deep, or bone invasion). The control group (Group 2) and sTGFbR2 + FGF21 treatment group (Group 4) showed the highest grade after 2 months of papain treatment, while the control group (Group 1) showed the lowest grade after 1 month of papain treatment. A slight decrease in grade was observed in the sTGFbR2 + αKlotho treatment group (Group 3) compared with the control group (Group 2) after 2 months of papain treatment.

[0117] OA stage was determined based on the total area of ​​cartilage affected by OA lesions, as shown in Figure 9. Among groups, the trend was similar to that seen for OA grade, but the lowest mean score for OA stage was observed in the sTGFbR2 + αKlotho-treated group (Group 3).

[0118] As shown in Figure 10, the OA score was determined by multiplying the grade and stage for the total OA value. Among groups, the trends were similar to those observed for OA stage. The group treated with papain for 2 months (Group 2) and the sTGFbR2 + FGF21 treatment group (Group 4) showed comparable highest scores. The group treated with papain for 1 month (Group 1) had a lower score compared with the group treated with papain for 2 months (Group 2). The sTGFbR2 + αKlotho treatment group (Group 3) showed the lowest score, indicating reduced lesion severity compared with the two control groups (Groups 1 and 2).

[0119] As shown in Figure 11, mild to moderate meniscal fibrosis was observed in the two control groups (Groups 1 and 2) and the sTGFbR2 + FGF21-treated group (Group 4), with Group 4 showing the highest meniscal fibrosis score. There was no meniscal fibrosis in the sTGFbR2 + αKlotho-treated group (Group 3).

[0120] Example 8 Comparison of treatments using a combination of αKlotho and sTGFbR2 independently First, we analyzed the combined effects of αKlotho ("K") and sTGFbR2 ("T"), identified as ("KT"), in an in vitro osteoarthritis model using high concentrations of TGFb1. Results analyzed by qPCR showed how the combination of both soluble factors synergistically supported the inhibition of hypertrophic markers and ECM protease inhibition when compared with treatment with a single factor (Figure 12A, K vs. T vs. KT). Accordingly, chondrocytes treated with both factors also showed higher protein expression of ACAN than αKlotho or sTGFbR2 (Figure 12B). Figure 12C illustrates the injection timeline.

[0121] Example 9 Embodiment

[0003] An embodiment of the present disclosure relates to a method for treating osteoarthritis in a subject in need thereof, comprising administering to the subject a first viral vector comprising a first nucleic acid sequence encoding a sTGFβ-R2 protein or an active fragment thereof and a second nucleic acid sequence encoding an αKlotho protein or an active fragment thereof, thereby treating osteoarthritis in the subject. According to one aspect, the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element. According to one aspect, the polycistronic element is an IRES or 2A sequence.

[0122]

[0003] Embodiments of the present disclosure relate to a method for treating osteoarthritis in a subject in need thereof, comprising administering to the subject a first viral vector comprising a first nucleic acid sequence encoding a sTGFβ-R2 protein or an active fragment thereof, and a second viral vector comprising a second nucleic acid sequence encoding an αKlotho protein or an active fragment thereof, thereby treating osteoarthritis in the subject. According to one aspect, the first nucleic acid sequence is operably linked to a first regulatory sequence, and / or the second nucleic acid sequence is operably linked to a second regulatory sequence. According to one aspect, the first regulatory sequence induces expression of the sTGFβ-R2 protein or an active fragment thereof, and / or the second regulatory sequence induces expression of the αKlotho protein or an active fragment thereof. According to one aspect, the first regulatory sequence and the second regulatory sequence each comprise a promoter. According to one aspect, the promoter is a constitutive promoter or an inducible promoter. According to one aspect, the first regulatory sequence and the second regulatory sequence each comprise a cell-specific promoter or a tissue-specific promoter. According to one embodiment, each of the first regulatory sequence and the second regulatory sequence comprises a liver-specific promoter. According to one embodiment, the regulatory sequence comprises a promoter selected from the group consisting of an hEf1α promoter, an shEf1α promoter (or a truncated hEf1α promoter), a CAG promoter (e.g., a cytomegalovirus, chicken β-actin intron, or rabbit β-globin gene splice acceptor), a CMV promoter, a hAAT promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, a thyroxine-binding globulin (TBG) promoter, a liver control region (HCR)-ApoCII hybrid promoter, a CASI promoter, a HCR-hAAT hybrid promoter, a hAAT promoter combined with a mouse albumin gene enhancer (Ealb) element, and an apolipoprotein E promoter. According to one embodiment, the first nucleic acid sequence is operably linked to a 3' untranslated region for RNA stability and expression in mammalian cells.According to one embodiment, the 3' untranslated region comprises a sequence selected from the group consisting of a WPRE sequence, a WPRE3 sequence, an SV40 late polyadenylation signal (e.g., a truncated version), an HBG polyadenylation signal, a rabbit β-globin polyadenylation signal, a bovine bgpA, an ETC polyadenylation signal, and any combination thereof. According to one embodiment, the first viral vector and / or the second viral vector is an adeno-associated viral (AAV) vector. According to one embodiment, the AAV vector is AAV-DJ. According to one embodiment, the AAV vector is derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, and AAVrh10.XX viral vectors. In one embodiment, the sTGFβ-R2 protein is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine sTGFβ-R2 proteins. In one embodiment, the sTGFβ-R2 protein is human sTGFβ-R2 protein. In one embodiment, the sTGFβ-R2 protein is canine sTGFβ-R2 protein. In one embodiment, the sTGFβ-R2 protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the αKlotho protein is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine αKlotho proteins. In one embodiment, the αKlotho protein is human αKlotho protein. In one embodiment, the αKlotho protein is canine αKlotho protein. According to one embodiment, the αKlotho protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. According to one embodiment, the sTGFβ-R2 protein and / or the αKlotho protein is an Fc fusion protein comprising an Ig Fc domain.According to one embodiment, the Ig Fc domain is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine Fc, or Fc subtypes including IgG1, IgG2a, IgG2b, IgG3, and IgG4. According to one embodiment, the Ig Fc domain comprises an amino acid sequence having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. According to one embodiment, the sTGFβ-R2 protein and / or the αKlotho protein are expressed and distributed systemically. According to one embodiment, the first viral vector and / or the second viral vector are administered by intravenous injection. According to one embodiment, the first viral vector and / or the second viral vector are administered by intra-articular injection into the cartilage at the site of osteoarthritis. According to one embodiment, the first viral vector and / or the second viral vector infects mesenchymal cells at the site of osteoarthritis. According to some embodiments, treating osteoarthritis in the subject comprises inhibiting the progression of osteoarthritis in the subject compared to a control subject. According to some embodiments, treating osteoarthritis in the subject comprises increasing, regenerating, or regrowing cartilage at the site of osteoarthritis in the subject compared to a control subject. According to some embodiments, treating osteoarthritis in the subject comprises inhibiting inflammation at the site of osteoarthritis in the subject compared to a control subject. According to some embodiments, the subject is a mammal. According to some embodiments, the mammal is a human. According to some embodiments, the mammal is a dog.

[0123]

[0003] Embodiments of the present disclosure relate to a method for treating osteoarthritis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination of αKlotho protein or an active fragment thereof and sTGFβ-R2 protein or an active fragment thereof, thereby treating osteoarthritis in the subject. According to one embodiment, the αKlotho protein or an active fragment thereof is administered as a soluble protein, and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein. According to one embodiment, the αKlotho protein and / or the sTGFβ-R2 protein are administered by intravenous injection. According to one embodiment, the αKlotho protein and / or the sTGFβ-R2 protein are administered by intra-articular injection into the cartilage at the site of osteoarthritis. According to one embodiment, treating osteoarthritis in the subject comprises inhibiting the progression of osteoarthritis in the subject compared to a control subject. According to certain aspects, treating osteoarthritis in the subject comprises increasing, regenerating, or regrowing cartilage at the site of osteoarthritis in the subject relative to a control subject. According to certain aspects, treating osteoarthritis in the subject comprises suppressing inflammation at the site of osteoarthritis in the subject relative to a control subject.

[0124]

[0003] Embodiments of the present disclosure relate to a method of treating osteoarthritis in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding a sTGFβ-R2 protein or an active fragment thereof, thereby treating osteoarthritis in the subject. According to one embodiment, the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element. According to one embodiment, the polycistronic element is an IRES or 2A sequence. According to one embodiment, the nucleic acid molecule is administered by intravenous injection. According to one embodiment, the nucleic acid molecule is administered by intra-articular injection into cartilage at the site of osteoarthritis. According to one embodiment, treating osteoarthritis in the subject comprises inhibiting the progression of osteoarthritis in the subject compared to a control subject. According to one embodiment, treating osteoarthritis in the subject comprises increasing, regenerating, or regrowing cartilage at the site of osteoarthritis in the subject compared to a control subject. In some embodiments, treating osteoarthritis in the subject comprises suppressing inflammation at the site of osteoarthritis in the subject compared to a control subject. In some embodiments, the nucleic acid molecule comprises DNA, RNA, or a combination thereof. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a dog.

[0125] Embodiments of the present disclosure relate to a vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble transforming growth factor β receptor II (sTGFβ-R2) protein or an active fragment thereof. In one embodiment, the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element. In one embodiment, the polycistronic element is an IRES or 2A sequence. In one embodiment, a first promoter is operably linked to the first nucleic acid sequence for expression of the αKlotho protein or an active fragment thereof in mammalian cells, and a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGFβ-R2 protein or an active fragment thereof in mammalian cells. In one embodiment, the first promoter and the second promoter are cell-specific or tissue-specific. In one embodiment, the first promoter and the second promoter are constitutive or inducible. According to one embodiment, the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine proteins. According to one embodiment, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins. According to one embodiment, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins. According to one embodiment, the αKlotho protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. According to one embodiment, the sTGFβ-R2 protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0126] An embodiment of the present disclosure relates to a pharmaceutical formulation comprising the above vector and a pharmaceutically acceptable excipient.

[0127] An embodiment of the present disclosure relates to a method for treating osteoarthritis in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a combination of αKlotho protein or an active fragment thereof and sTGFβ-R2 protein or an active fragment thereof at a site exhibiting osteoarthritis in the mammal, wherein the method results in inhibiting the progression of the osteoarthritis compared to an untreated state, increasing, regenerating, or regrowing cartilage at the osteoarthritis site compared to an untreated state, or inhibiting inflammation compared to an untreated state. In one embodiment, the mammal is a dog or a human. In one embodiment, the αKlotho protein or an active fragment thereof is administered as a soluble protein, and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein. In one embodiment, the αKlotho protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection, and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection. According to one embodiment, a vector comprising a first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof is administered, wherein the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof. According to one embodiment, a vector comprising the first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and the second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof is administered by intra-articular cartilage injection, wherein the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof.According to one embodiment, a first vector comprising a first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and a second vector comprising a second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof are administered, and the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof. According to one embodiment, a first vector comprising a first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and a second vector comprising a second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof are administered by intra-articular cartilage injection, and the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof. According to one embodiment, the vector is a recombinant virus. According to one embodiment, the vector is a parvovirus. According to one embodiment, the vector is an AAV vector. In one embodiment, the AAV vector is AAV-DJ. In one embodiment, the vector is an AAV vector classified as serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrh10.XX, or a combination thereof. In one embodiment, the vector infects mesenchymal cells at the osteoarthritis site. In one embodiment, the first vector and the second vector are recombinant viruses. In one embodiment, the first vector and the second vector are parvoviruses. In one embodiment, the first vector and the second vector are AAV vectors. In one embodiment, the first vector and the second vector are AAV-DJ vectors. According to one aspect, the first vector and the second vector are AAV vectors classified into the serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrh10.XX, or a combination thereof.According to one embodiment, the first vector and the second vector infect mesenchymal cells at the osteoarthritis site. According to one embodiment, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins. According to one embodiment, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins. According to one embodiment, the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine proteins. According to one embodiment, the αKlotho protein has 90% or more sequence identity to the amino acid sequence of the αKlotho protein corresponding to SEQ ID NO: 1. According to one embodiment, the sTGFβ-R2 protein has 90% or more sequence identity to the amino acid sequence of the sTGFβ-R2 protein corresponding to SEQ ID NO: 3.

[0128] Embodiments of the present disclosure relate to a vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble transforming growth factor beta receptor II (sTGFβ-R2) protein or an active fragment thereof. In one aspect, a first promoter is operably linked to the first nucleic acid sequence for expression of the αKlotho protein or an active fragment thereof in a mammalian cell, and a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGFβ-R2 protein or an active fragment thereof in a mammalian cell. In one aspect, the first promoter and the second promoter are cell-specific or tissue-specific. In one aspect, the first promoter and the second promoter are constitutive or inducible. In one aspect, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins. In one aspect, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins. According to one embodiment, the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine proteins. According to one embodiment, the αKlotho protein has 90% or more sequence identity to the amino acid sequence of the αKlotho protein corresponding to SEQ ID NO: 1. According to one embodiment, the sTGFβ-R2 protein has 90% or more sequence identity to the amino acid sequence of the sTGFβ-R2 protein corresponding to SEQ ID NO: 3.

[0129] An embodiment of the present disclosure relates to a pharmaceutical formulation comprising the above vector in a pharmaceutically acceptable excipient.

[0130] All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0131] While various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention.

[0132] Sequence Listing [Table 1]

[0133] [Table 2]

[0134] [Table 3]

[0135] [Table 4]

[0136] [Table 5]

[0137] [Table 6]

[0138] [Table 7]

[0139] [Table 8]

[0140]

Table 9

[0141]

Table 10

[0142]

Table 11

Claims

1. 1. A method of treating osteoarthritis in a subject in need thereof, comprising: a first nucleic acid sequence encoding a sTGFβ-R2 protein or an active fragment thereof; and A second nucleic acid sequence encoding the αKlotho protein or an active fragment thereof. administering to the subject a first viral vector comprising: thereby treating osteoarthritis in said subject. The method.

2. 2. The method of claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element.

3. The method of claim 2, wherein the polycistronic element is an IRES or 2A sequence.

4. 1. A method of treating osteoarthritis in a subject in need thereof, comprising: a first viral vector comprising a first nucleic acid sequence encoding a sTGFβ-R2 protein or an active fragment thereof; and A second viral vector comprising a second nucleic acid sequence encoding the αKlotho protein or an active fragment thereof. to said subject, thereby treating osteoarthritis in said subject. The method.

5. 5. The method of any one of claims 1 to 4, wherein the first nucleic acid sequence is operably linked to a first regulatory sequence and / or the second nucleic acid sequence is operably linked to a second regulatory sequence.

6. The method of claim 5, wherein the first regulatory sequence causes expression of the sTGFβ-R2 protein or an active fragment thereof, and / or the second regulatory sequence causes expression of the αKlotho protein or an active fragment thereof.

7. 7. The method of claim 5 or claim 6, wherein each of the first regulatory sequence and the second regulatory sequence comprises a promoter.

8. The method of claim 7 , wherein the promoter is a constitutive promoter or an inducible promoter.

9. The method of any one of claims 5 to 8, wherein each of the first regulatory sequence and the second regulatory sequence comprises a cell-specific promoter or a tissue-specific promoter.

10. The method of any one of claims 5 to 9, wherein the first regulatory sequence and the second regulatory sequence each comprise a liver-specific promoter.

11. 10. The method of any one of claims 5 to 9, wherein the regulatory sequence comprises a promoter selected from the group consisting of an hEf1α promoter, an shEf1α promoter (or a truncated hEf1α promoter), a CAG promoter (e.g., a splice acceptor of a cytomegalovirus, a chicken β-actin intron, or a rabbit β-globin gene), a CMV promoter, a hAAT promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, a thyroxine-binding globulin (TBG) promoter, a liver control region (HCR)-ApoCII hybrid promoter, a CASI promoter, a HCR-hAAT hybrid promoter, a hAAT promoter in combination with a mouse albumin gene enhancer (Ealb) element, and an apolipoprotein E promoter.

12. 12. The method of any one of claims 1 to 11, wherein the first nucleic acid sequence is operably linked to a 3' untranslated region for RNA stability and expression in mammalian cells.

13. 13. The method of claim 12, wherein the 3' untranslated region comprises a sequence selected from the group consisting of a WPRE sequence, a WPRE3 sequence, an SV40 late polyadenylation signal (e.g., a truncated version), an HBG polyadenylation signal, a rabbit β-globin polyadenylation signal, a bovine bgpA, an ETC polyadenylation signal, and any combination thereof.

14. The method according to any one of claims 1 to 13, wherein the first viral vector and / or the second viral vector is an adeno-associated viral (AAV) vector.

15. The method of claim 14, wherein the AAV vector is AAV-DJ.

16. 15. The method of claim 14, wherein the AAV vector is derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, and AAVrhlO.XX viral vectors.

17. 17. The method of any one of claims 1 to 16, wherein the sTGFβ-R2 protein is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine sTGFβ-R2 proteins.

18. The method according to any one of claims 1 to 17, wherein the sTGFβ-R2 protein is a human sTGFβ-R2 protein.

19. The method according to any one of claims 1 to 18, wherein the sTGFβ-R2 protein is a canine sTGFβ-R2 protein.

20. The method according to any one of claims 1 to 19, wherein the sTGFβ-R2 protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

3.

21. 21. The method of any one of claims 1 to 20, wherein the αKlotho protein is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine αKlotho proteins.

22. The method according to any one of claims 1 to 21, wherein the αKlotho protein is a human sTGFβ-R2 protein.

23. The method of any one of claims 1 to 22, wherein the αKlotho protein is a canine sTGFβ-R2 protein.

24. The method of any one of claims 1 to 23, wherein the αKlotho protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:

1.

25. The method according to any one of claims 1 to 24, wherein the sTGFβ-R2 protein and / or the αKlotho protein is an Fc fusion protein comprising an Ig Fc domain.

26. 26. The method of claim 25, wherein the Ig Fc domain is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine Fc, or subtypes of the Fc, including IgG1, IgG2a, IgG2b, IgG3, and IgG4.

27. 27. The method of claim 25 or claim 26, wherein the Ig Fc domain comprises an amino acid sequence having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:

7.

28. The method of any one of claims 1 to 27, wherein the sTGFβ-R2 protein and / or the αKlotho protein are expressed and distributed systemically.

29. The method of any one of claims 1 to 28, wherein the first viral vector and / or the second viral vector is administered by intravenous injection.

30. 30. The method of any one of claims 1 to 29, wherein the first viral vector and / or the second viral vector is administered by intra-articular injection into the cartilage at the site of osteoarthritis.

31. The method of any one of claims 1 to 30, wherein the first viral vector and / or the second viral vector infects mesenchymal cells at the site of osteoarthritis.

32. 32. The method of any one of claims 1 to 31, wherein treating osteoarthritis in the subject comprises inhibiting the progression of osteoarthritis in the subject compared to a control subject.

33. 33. The method of any one of claims 1 to 32, wherein treating osteoarthritis in the subject comprises increasing, regenerating, or regrowing cartilage at sites of osteoarthritis in the subject compared to a control subject.

34. 34. The method of any one of claims 1 to 33, wherein treating osteoarthritis in the subject comprises suppressing inflammation at sites of osteoarthritis in the subject compared to a control subject.

35. The method of any one of claims 1 to 34, wherein the subject is a mammal.

36. 36. The method of claim 35, wherein the mammal is a human.

37. 36. The method of claim 35, wherein the mammal is a dog.

38. 1. A method of treating osteoarthritis in a subject in need thereof, comprising: an αKlotho protein or an active fragment thereof; sTGFβ-R2 protein or an active fragment thereof administering to said subject a therapeutically effective amount of a combination of thereby treating osteoarthritis in said subject. The method.

39. 39. The method of claim 38, wherein the αKlotho protein or an active fragment thereof is administered as a soluble protein, and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein.

40. 40. The method of claim 38 or 39, wherein the αKlotho protein and / or the sTGFβ-R2 protein is administered by intravenous injection.

41. 41. The method of any one of claims 38 to 40, wherein the αKlotho protein and / or the sTGFβ-R2 protein is administered by intra-articular injection into the cartilage at the site of osteoarthritis.

42. 42. The method of any one of claims 38 to 41, wherein treating osteoarthritis in the subject comprises inhibiting the progression of osteoarthritis in the subject compared to a control subject.

43. 43. The method of any one of claims 38 to 42, wherein treating osteoarthritis in the subject comprises increasing, regenerating, or regrowing cartilage at the site of osteoarthritis in the subject compared to a control subject.

44. 44. The method of any one of claims 38 to 43, wherein treating osteoarthritis in the subject comprises suppressing inflammation at sites of osteoarthritis in the subject compared to a control subject.

45. 1. A method of treating osteoarthritis in a subject in need thereof, comprising: a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof; and A second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof. administering to the subject a nucleic acid molecule comprising thereby treating osteoarthritis in said subject. The method.

46. 46. ​​The method of claim 45, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element.

47. 47. The method of claim 46, wherein the polycistronic element is an IRES or 2A sequence.

48. 48. The method of any one of claims 45 to 47, wherein the nucleic acid molecule is administered by intravenous injection.

49. 48. The method of any one of claims 45 to 47, wherein the nucleic acid molecule is administered by intra-articular injection into the cartilage at the site of osteoarthritis.

50. 50. The method of any one of claims 45 to 49, wherein treating osteoarthritis in the subject comprises inhibiting the progression of osteoarthritis in the subject compared to a control subject.

51. 51. The method of any one of claims 45 to 50, wherein treating osteoarthritis in the subject comprises increasing, regenerating, or regrowing cartilage at sites of osteoarthritis in the subject compared to a control subject.

52. 52. The method of any one of claims 45 to 51, wherein treating osteoarthritis in the subject comprises suppressing inflammation at sites of osteoarthritis in the subject compared to a control subject.

53. 53. The method of any one of claims 45 to 52, wherein the nucleic acid molecule comprises DNA, RNA, or a combination thereof.

54. The method of any one of claims 45 to 53, wherein the subject is a mammal.

55. 55. The method of claim 54, wherein the mammal is a human.

56. 55. The method of claim 54, wherein the mammal is a dog.

57. a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof; and A second nucleic acid sequence encoding a soluble transforming growth factor beta receptor II (sTGFβ-R2) protein or an active fragment thereof. A vector comprising:

58. 58. The vector of claim 57, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element.

59. 59. The vector of claim 58, wherein the polycistronic element is an IRES or 2A sequence.

60. 60. The vector of claim 58 or claim 59, wherein a first promoter is operably linked to the first nucleic acid sequence for expression of the αKlotho protein or an active fragment thereof in mammalian cells, and a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGFβ-R2 protein or an active fragment thereof in mammalian cells.

61. 61. The vector of claim 60, wherein the first promoter and the second promoter are cell-specific or tissue-specific.

62. 62. The vector of claim 60 or claim 61, wherein the first promoter and the second promoter are constitutive or inducible.

63. 63. The vector of any one of claims 57 to 62, wherein the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine proteins.

64. The vector according to any one of claims 57 to 63, wherein the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins.

65. The vector according to any one of claims 57 to 63, wherein the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins.

66. The vector of any one of claims 57 to 65, wherein the αKlotho protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:

1.

67. The vector according to any one of claims 57 to 66, wherein the sTGFβ-R2 protein comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

3.

68. A pharmaceutical formulation comprising a vector according to any one of claims 57 to 67 and a pharmaceutically acceptable excipient.

69. 1. A method of treating osteoarthritis in a mammal in need thereof, comprising: administering a therapeutically effective amount of a combination of αKlotho protein or an active fragment thereof and sTGFβ-R2 protein or an active fragment thereof to said mammal at a site exhibiting osteoarthritis in said mammal; The progression of the osteoarthritis is inhibited compared to an untreated state, or an increase, regeneration, or regrowth of cartilage at the site of said osteoarthritis compared to an untreated condition; or Reduced inflammation compared to untreated The method.

70. 70. The method of claim 69, wherein the mammal is a dog or a human.

71. 70. The method of claim 69, wherein the αKlotho protein or an active fragment thereof is administered as a soluble protein and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein.

72. 70. The method of claim 69, wherein the αKlotho protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection, and the sTGFβ-R2 protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection.

73. 70. The method of claim 69, wherein a vector comprising a first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof is administered, wherein the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof.

74. 70. The method of claim 69, wherein a vector comprising a first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof is administered by intra-articular cartilage injection, wherein the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof.

75. 70. The method of claim 69, wherein a first vector comprising a first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and a second vector comprising a second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof are administered, wherein the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof.

76. 70. The method of claim 69, wherein a first vector comprising a first nucleic acid sequence encoding the αKlotho protein or an active fragment thereof and a second vector comprising a second nucleic acid sequence encoding the sTGFβ-R2 protein or an active fragment thereof are administered by intra-articular cartilage injection, wherein the first nucleic acid sequence is expressed to produce the αKlotho protein or an active fragment thereof, and the second nucleic acid sequence is expressed to produce the sTGFβ-R2 protein or an active fragment thereof.

77. 75. The method of claims 73 and 74, wherein the vector is a recombinant virus.

78. The method of claims 73 and 74, wherein the vector is a parvovirus.

79. The method of claims 73 and 74, wherein the vector is an AAV vector.

80. The method of claims 73 and 74, wherein the AAV vector is AAV-DJ.

81. 75. The method of claims 73 and 74, wherein the vector is an AAV vector classified as serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrhlO.XX, or a combination thereof.

82. 75. The method of claims 73 and 74, wherein the vector infects mesenchymal cells at the site of osteoarthritis.

83. 77. The method of claims 75 and 76, wherein the first vector and the second vector are recombinant viruses.

84. 77. The method of claims 75 and 76, wherein the first vector and the second vector are parvoviruses.

85. 77. The method of claims 75 and 76, wherein the first vector and the second vector are AAV vectors.

86. The method of claims 75 and 76, wherein the first vector and the second vector are AAV-DJ vectors.

87. 77. The method of claims 75 and 76, wherein the first vector and the second vector are AAV vectors classified into serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrhlO.XX, or a combination thereof.

88. 77. The method of claims 75 and 76, wherein the first vector and the second vector infect mesenchymal cells at the site of osteoarthritis.

89. 70. The method of claim 69, wherein the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins.

90. 70. The method of claim 69, wherein the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins.

91. 70. The method of claim 69, wherein the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine proteins.

92. 70. The method of claim 69, wherein the αKlotho protein has 90% or more sequence identity to the amino acid sequence of the αKlotho protein corresponding to SEQ ID NO:

1.

93. 70. The method of claim 69, wherein the sTGFβ-R2 protein has 90% or greater sequence identity to the amino acid sequence of the sTGFβ-R2 protein corresponding to SEQ ID NO:

3.

94. A vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble transforming growth factor beta receptor II (sTGFβ-R2) protein or an active fragment thereof.

95. 95. The vector of claim 94, wherein a first promoter is operably linked to the first nucleic acid sequence for expression of the αKlotho protein or an active fragment thereof in a mammalian cell, and a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGFβ-R2 protein or an active fragment thereof in a mammalian cell.

96. 96. The vector of claim 95, wherein the first promoter and the second promoter are cell-specific or tissue-specific.

97. 96. The vector of claim 95, wherein the first promoter and the second promoter are constitutive or inducible.

98. 95. The vector of claim 94, wherein the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins.

99. 95. The vector of claim 94, wherein the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins.

100. 95. The vector of claim 94, wherein the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine proteins.

101. 95. The vector of claim 94, wherein the αKlotho protein has 90% or more sequence identity to the amino acid sequence of the αKlotho protein corresponding to SEQ ID NO:

1.

102. 95. The vector of claim 94, wherein the sTGFβ-R2 protein has 90% or more sequence identity to the amino acid sequence of the sTGFβ-R2 protein corresponding to SEQ ID NO:

3.

103. 95. A pharmaceutical formulation comprising the vector of claim 94 in a pharmaceutically acceptable inactive ingredient.

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