Method for treating osteoarthritis

Abstract

A method for treating osteoarthritis is provided by administering αKlotho protein and sTGFβ-R2 protein to a site of a mammal showing symptoms of osteoarthritis, such as a knee joint. Both αKlotho protein and sTGFβ-R2 protein are present at the site of osteoarthritis.

Description

Background Art

[0001] Articular cartilage is a tissue that undergoes considerable 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. Ageing and the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 2016 Jul;12(7):412 - 20). As a result, articular cartilage contributes to the onset of osteoarthritis (OA) by causing a decline in homeostasis and limitations in repair ability (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 the elderly and is a major cause of physical 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 analgesia 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 still no available treatment to cure this condition.

[0002] The lack of an effective clinical treatment for osteoarthritis supports the worldwide increase 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 has several limitations, including the need to harvest healthy donor cartilage by an independent surgical procedure, the limited proliferative capacity of primary chondrocytes, and the difficulty of treating large-scale defects.

[0003] Therefore, there remains a need to find an effective treatment that can avoid surgical procedures and treat this condition, which is associated with both aging and joint trauma. There is also a need to develop therapeutic targets for improving the low regenerative capacity seen in adults and that worsens with aging, and for assisting in the restoration of 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 of treatment by administering to the mammal, at a site in the mammal exhibiting osteoarthritis, a therapeutically effective amount of an αKlotho protein or an active fragment thereof, and a therapeutically effective amount of an sTGFβ-R2 protein or an active fragment thereof, or a combination thereof, wherein the progression of the osteoarthritis is suppressed compared to an untreated state, cartilage at the site of the osteoarthritis increases, regenerates, or regrows compared to an untreated state, or inflammation is suppressed compared to an untreated state. The site of osteoarthritis is a site exhibiting symptoms of osteoarthritis. Osteoarthritis is the most common form of arthritis, and millions of people worldwide are affected by osteoarthritis. Osteoarthritis occurs when the protective cartilage that cushions the ends of bones wears away over time. Osteoarthritis can damage any joint, but this disorder most commonly affects the joints of the hands, knees, lower back, and spine. Symptoms of osteoarthritis include pain, stiffness, tenderness, loss of flexibility, grating, bone spurs, and swelling.

[0005] The functional proteins described herein can be full-length proteins or proteins that differ from the full-length proteins but retain all or part of the activity of the full-length proteins.

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

[0007] This patent or patent application file contains color drawings. Copies of this patent or patent application publication, which include color drawings, will be provided by the United States Patent and Trademark Office upon request and payment of the required fees. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0008]

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BRIEF DESCRIPTION OF THE INVENTION

[0009] The present disclosure provides a method and composition for treating or preventing osteoarthritis, such as osteoarthritis occurring in articular cartilage, using a therapeutically effective amount of a combination of an αKlotho protein or an active fragment thereof and a soluble tumor growth factor β receptor 2 (sTGFβR2) protein or an active fragment thereof, which are administered as soluble proteins to a mammal in need of treatment or prevention, or a vector used to express the soluble protein at a site indicating osteoarthritis in the mammal. According to one aspect, soluble tumor growth factor β receptor 2 (sTGFβR2) is administered together with the αKlotho protein to treat or prevent osteoarthritis in a joint such as the knee where articular cartilage is present. According to one aspect, soluble tumor growth factor β receptor 2 (sTGFβR2) and αKlotho are administered separately or simultaneously such that both are present at the administration site. According to one aspect, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are encoded by a plurality of nucleic acids contained within one or more vectors or are combined within a single viral vector such as AAV and are administered to treat or prevent osteoarthritis and / or a disease or symptom associated with osteoarthritis.

[0010] According to one aspect, sTGFβR2 acts to inhibit TGFβ1, thereby suppressing osteophyte formation despite increasing 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, TGFβ1 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-beta / 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, chondrocyte hypertrophy is significantly increased by an increase in the ALK1 receptor / ALK5 receptor ratio (see Blaney Davidson EN, Remst DFG, Vitters EL, van Beuningen HM, Blom AB, Goumans M-J, 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 by long-term exposure to TGF-β1 (see Pohlers D, Beyer A, Koczan D, Wilhelm T, Thiesen H-J, 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 aspect, soluble TGFβR2 (sTGFβR2), which lacks a membrane-binding domain and has a 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), can then modulate the effects of TGF-β1 in joints.

[0012] According to one aspect, αKlotho suppresses or prevents the degradation of the extracellular matrix (ECM) (see Chuchana P, Mausset-Bonnefont A-L, 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 between aging and osteoarthritis (see Pasztói M, Nagy G, Geher P, Lakatos T, Tóth 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 (see 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 controls surface glycoproteins such as ion channels, insulin-like growth factor 1 (IGF-1) / insulin, and Wnt by removing terminal sialic acid from N-linked glycans (see Dalton GD, Xie J, An S-W, Huang C-L. 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 aspect, a method for treating osteoarthritis in a mammal in need of treatment, comprising administering a therapeutically effective amount of a combination of an αKlotho protein or an active fragment thereof and an sTGFβ-R2 protein or an active fragment thereof to the mammal at a site exhibiting osteoarthritis in the mammal, wherein the progression of the osteoarthritis is suppressed compared to the untreated state, cartilage in the site of the osteoarthritis increases, regenerates, or regrows compared to the untreated state, or inflammation is suppressed compared to the untreated state. According to one aspect, the mammal is a dog or a human. According to one aspect, 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 aspect, the αKlotho protein or an active fragment thereof is administered by intra-articular cartilage injection as a soluble protein, and the sTGFβ-R2 protein or an active fragment thereof is administered by intra-articular cartilage injection as a soluble protein. According to one aspect, a vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding an sTGFβ-R2 protein or an active fragment thereof is administered, 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 aspect, a vector comprising a first nucleic acid sequence encoding an αKlotho protein or an active fragment thereof and a second nucleic acid sequence encoding an sTGFβ-R2 protein or an active fragment thereof is administered by intra-articular cartilage injection, 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 aspect, 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 an sTGFβ-R2 protein or an active fragment thereof are administered, 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 aspect, 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 an sTGFβ-R2 protein or an active fragment thereof are administered by intra-articular cartilage injection, 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 aspect, the vector is a recombinant virus. According to one aspect, the vector is a parvovirus. According to one aspect, the vector is an AAV vector. According to one aspect, the AAV vector is AAV-DJ. According to one aspect, the vector is an AAV vector classified into 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, an example of which is AAVrh10.32), or a combination thereof. According to one aspect, the vector infects mesenchymal cells at the site of osteoarthritis. According to one aspect, the first vector and the second vector are recombinant viruses. According to one aspect, the first vector and the second vector are parvoviruses. According to one aspect, the first vector and the second vector are AAV vectors. According to one aspect, 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 a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrh10.XX (where xx represents various known variants), or a combination thereof. According to one aspect, the first vector and the second vector infect mesenchymal cells at the site of osteoarthritis. According to one aspect, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins.According to certain embodiments, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins. According to certain embodiments, 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 certain embodiments, the αKlotho protein has an amino acid sequence of the αKlotho protein corresponding to SEQ ID NO: 1 below.

Chem.

[0014] According to certain embodiments, the nucleic acid sequence encoding the αKlotho protein is the nucleic acid sequence encoding the αKlotho protein corresponding to SEQ ID NO: 2 below

Chem.

Chem.

Chem.

[0015] According to certain embodiments, the sTGFβ-R2 protein has the amino acid sequence of the sTGFβR2 receptor protein corresponding to SEQ ID NO: 3 below (the sTGFbR2 amino acid sequence is the sequence in bold in the IGG FC domain, and MGRGLLRGLWPLHIVLWTRIAST is the secretion signal)

Chemical formula

[0016] According to certain embodiments, 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

Chemical formula

[0017] According to certain embodiments, the sTGFβ-R2 protein and / or the αKlotho protein is an Fc fusion protein comprising an Ig Fc domain. According to certain embodiments, the Ig Fc domain is selected from the group consisting of the Fc of human, dog, cat, cow, sheep, goat, horse, mouse, and pig, or subtypes of said Fc including IgG1, IgG2a, IgG2b, IgG3, and IgG4.

[0018] According to certain embodiments, the Ig Fc domain has an amino acid sequence corresponding to SEQ ID NO: 5 below

Chemical formula

[0019] According to certain embodiments, the Ig Fc domain has an amino acid sequence corresponding to SEQ ID NO: 6 below

Chemical formula

[0020] According to one aspect, the Ig Fc domain has an amino acid sequence corresponding to SEQ ID NO: 7 below

Chemical formula

[0021] According to one aspect, an amino acid sequence having the homology described for the αKlotho protein amino acid sequence or the sTGFβ-R2 protein amino acid sequence can be determined by obtaining the crystal structure of the αKlotho protein or the 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. The portions of the protein identified as being inactive are suitable for amino acid substitutions, modifications, or mutations to create a protein having the claimed homology. Also, the active portions may be modified, substituted, or mutated as long as useful binding or activity results. An Ig Fc sequence 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 of skill in the art, and those methods include the general methods described in Newcomer et al., PNAS, Vol. 90, pp. 9223-9227 (October 1993), and those of skill in the art can use or modify those methods to determine the binding site for the αKlotho protein or the sTGFβ-R2 protein. Software programs such as MED-SuMO (distributed by MEDIT), TRAPP (Max Planck Institute of Biophysics, Molecular Cell Modeling Group, 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) can be used to determine 3D structures, binding pockets, tunnels and channels, surface properties and cavities, and ligand binding sites, etc.Useful databases include sc-PDB (Strasbourg University), CASTp, Pocketome (encyclopedia of the three-dimensional structure ensemble of druggable binding sites that can be experimentally identified from co-crystal structures in the Protein Data Bank), motifs and sites of PDBe, LigASite, PROtein SURFace ExploreR, 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 2, Italy), IsoMIF (University of Sherbrooke, Canada), LISE (Institute of Biomedical Sciences, Academia Sinica), SiteHound-web (Sanchez Laboratory, Mount Sinai School of Medicine, New York), and MultiBind (Bioinformatics Group, Tel Aviv University).

[0022] Aspects of the present disclosure provide 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. According to certain aspects, 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. According to certain aspects, the first promoter and the second promoter are cell-specific or tissue-specific. According to certain aspects, the first promoter and the second promoter are constitutive or inducible.

[0023] The present disclosure provides a pharmaceutical preparation comprising a vector containing 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 a pharmaceutically acceptable excipient.

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

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

[0026] In referring to the present disclosure, technical and scientific terms used in the descriptions herein have the meanings 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, references to "protein" include more than one protein, and references to "excipient" include more than one excipient.

[0028] It is further to be understood that the use of "or" means "and / or" unless specifically indicated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes", and "including" are interchangeable and not intended to be limiting. Also, when the term "comprising" is used in the description of various embodiments, those of ordinary skill in the art will understand that in certain instances, an embodiment may alternatively be described using the phrases "consisting essentially of" or "consisting of".

[0029] As used herein, "gene" refers to a nucleic acid region that expresses a polynucleotide such as RNA, and is also referred to as a transcription region. The transcribed polynucleotide may have a sequence that encodes a polypeptide such as a functional protein, and that sequence can be translated into the encoded polypeptide when placed under the control of an appropriate control region. A gene can include several fragments, such as a promoter, a 5' leader sequence, a coding sequence, and a 3' untranslated sequence such as a polyadenylation site, which are operably linked. A chimeric gene or recombinant gene is a gene not normally found in nature, for example, a gene in which a promoter is not part or all of the DNA region it is normally associated with. "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 introducing recombinant DNA or foreign DNA into a host cell. The introduced DNA may remain unintegrated or is preferably integrated into the genome of the host cell. Gene delivery can occur, for example, by transduction using a viral vector or by transformation of cells using known methods such as the electroporation method, cell bombardment, etc.

[0031] "Transgene" refers to a gene introduced into a host cell. The transgene may include a sequence specific to the cell, a sequence not naturally present in the cell, or a combination thereof. The transgene may include a sequence encoding one or more proteins, and those sequences may be operably linked to appropriate control sequences for expression of the coding sequence 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 rAAV. For example, transduction of a target cell by an rAAV virion results in the introduction of the rAAV vector contained in that virion into the transduced cell. "Host cell" or "target cell" refers to the cell into which nucleic acid delivery occurs.

[0033] Examples of "functional proteins" include variants, mutants, homologs, and functional fragments of full-length proteins. Those skilled in the art will be able to readily construct proteins homologous to full-length proteins that wholly or partially retain the activity of the full-length proteins based on the present disclosure.

[0034] "Vector" generally refers to a nucleic acid construct suitable for cloning and expressing nucleotide sequences. An example of a vector is a viral vector. The term "vector" may also refer to a transport vehicle, such as a virus or virion, including vectors that can be introduced into host cells 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, AAVrh10.XX, etc. The rAAV vector may have one or preferably all wild-type AAV genes deleted, but still contains a functional ITR nucleic acid sequence. The functional ITR sequence is necessary for replication, rescue, and packaging of the AAV virion. The TR sequence may be a wild-type sequence, or a sequence that is substantially identical (as defined below), or may be modified, for example, by nucleotide insertion, mutation, deletion, or substitution, as long as these sequences are functional.

[0036] "Therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result, such as the result for osteoarthritis and related diseases or conditions, at the required dosage and for the required period. The therapeutically effective amount of parvovirus virions or pharmaceutical compositions may vary depending on factors such as the disease state, age, gender, and weight of the individual, as well as the ability of the parvovirus virions or pharmaceutical compositions to induce a desired response in the individual. The dosing regimen may be adjusted to achieve an optimal therapeutic response. Also, a therapeutically effective amount is usually an amount that exceeds the toxic or harmful effects of the parvovirus virions or pharmaceutical compositions, if any, of the treatment.

[0037] "Prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic outcome, such as the prevention or suppression of osteoarthritis, at the required dosage and for the required period. The prophylactic dosage may be used in a subject before or at an early stage of the 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 herein with "nucleic acid". The nucleic acid may be an oligonucleotide or a polynucleotide. The nucleic acid may be DNA or RNA. The nucleic acid may be a gene. The nucleic acid may be chemically modified or may be an artificial construct. Artificial nucleic acids include peptide nucleic acid (PNA), morpholino nucleic acid, and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from natural DNA or RNA by a modification to the molecular backbone. Also, phosphorothioate nucleotides may be used.

[0039] As used herein, a "nucleic acid construct" is understood to mean an artificial nucleic acid molecule generated using recombinant DNA technology. A nucleic acid construct is a single-stranded or double-stranded nucleic acid molecule that has been modified to contain segments of nucleic acids that are combined and juxtaposed in a way that does not occur naturally. 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 nucleotide sequences that can effect gene expression in a host cell or host organism that is compatible with such nucleotide sequences. An expression cassette or expression vector typically contains at least appropriate transcriptional control sequences and optionally a 3' transcription termination signal. Additional factors necessary or beneficial for effecting 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. When increasing the expression of a target protein, the nucleic acid construct may be an mRNA or analog thereof that can be recognized by a cell or more specifically a ribosome to produce multiple copies of the protein. When suppressing the expression of a target sequence, the RNA may be in a form that acts by preventing the production of protein by ribosomes. This can be accomplished by the mechanism of RNAi, shRNA, miRNA, or Pri-miRNA. According to certain embodiments, the target sequence can be increased by suppression via delivery of an mRNA (or analog) or shRNA (or analog) that controls the target sequence by suppressing a known suppressor of the target sequence. This can also be accomplished 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 that are in a functional relationship. Nucleic acids are "operably linked" when they are in a functional relationship with another nucleic acid sequence. For example, if a transcriptional control sequence affects the transcription of a coding sequence, then that transcriptional regulatory sequence is operably linked to that coding sequence. By "operably linked" is meant that the DNA sequences being linked are usually contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

[0041] "Expression control sequence" refers to a nucleic acid sequence that controls the expression of a nucleotide sequence to which it is operably linked. When an expression regulatory sequence regulates and controls the transcription and / or translation of a nucleotide sequence, that expression regulatory sequence is "operably linked" to that nucleotide sequence. Thus, expression regulatory sequences can include promoters, enhancers, internal ribosome entry sites (IRES) within the sequence, transcription terminators, start codons preceding protein-coding genes, splicing signals for introns, 2A peptide sequences (which enable polycistronic expression), and stop codons. According to one aspect, the first nucleic acid sequence and the second nucleic acid sequence encoding sTGFβ-R2 protein and αKlotho are separated by a polycistronic element. A polycistronic element is 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 regulatory sequence" is intended to include at least sequences designed such that their presence affects expression, and may also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression regulatory sequences. The term can also include the design of nucleic acid sequences such that unwanted potential start codons, whether in-frame or out-of-frame, are removed from the sequence. The design of nucleic acid sequences such that unwanted potential splice sites are removed can also be included in expression regulatory sequences. Expression regulatory sequences also include sequences that add a polyA tail, sometimes called a polyA sequence (i.e., a chain of adenine residues at the 3' end of mRNA), i.e., a polyadenylation sequence (pA). Expression regulatory sequences may also be designed to confer mRNA stability. Expression regulatory sequences that affect the stability of transcription and translation, such as promoters, and sequences that result in appropriate translation for use in insect cells, such as Kozak sequences, are well known to those skilled in the art. Expression regulatory sequences can have the property of regulating the nucleotide sequence to which they are operably linked such that a low or high expression level is achieved.

[0042] It is also possible to fuse a functional domain to a known protein. This also applies when a mitochondrial signal is fused to CAT (catalase) such that the catalase is targeted to be transported to the mitochondria and catalase performs its function inside or in the vicinity of the mitochondria instead of its natural location. It is possible to add targeting signals to other proteins to target them to other parts of the cell, or to secrete those proteins from the cell. In the case of some proteins, the natural sequence can be replaced with a better-known version for improved effect, such as selecting a human or mouse secretion signal for TGFbR2 and fusing it to a canine-type protein.

[0043] A "promoter" or "transcription control sequence" functions to regulate the transcription of one or more coding sequences, is located upstream relative to the direction of transcription at the transcription start site of the coding sequence, and includes a binding site for DNA-dependent RNA polymerase, a transcription start site, and, without limitation, 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., including attenuators or enhancers, and silencers). It refers to a nucleic acid fragment structurally defined by the presence of any other DNA sequence. 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 the application of a chemical inducer. A "tissue-specific" promoter is active only in specific types of tissues or cells. The present disclosure provides an operable linkage of a nucleic acid construct to a mammalian cell-compatible expression regulatory sequence (e.g., a promoter). Many such promoters are known in the art (see Sambrook and Russell, 2001, supra). Constitutive promoters that are widely 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 result in effective constitutive expression are also disclosed. Inducible, tissue-specific, cell type-specific, or cell cycle-specific promoters are disclosed. In the disclosed embodiments, a nucleotide sequence encoding porphobilinogen deaminase is operably linked to a liver-specific promoter. The liver-specific promoter is particularly preferred for use in combination with non-erythroid deaminase.Preferably, in the constructs of the present disclosure, the expression control sequences for liver-specific expression are selected from the group consisting of, for example, the α1-antitrypsin (AAT) promoter, the thyroxine-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 neuroblastoma-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] The "3'UTR" or "3' untranslated sequence" (often also called the 3' untranslated region or 3' end) refers to a nucleic acid sequence found downstream of the coding sequence of a gene and includes, for example, the transcription termination site and (although not all but most eukaryotic mRNAs) the polyadenylation signal (such as AAUAAA or its variants, etc.). After transcription termination, the mRNA transcript may be cleaved downstream of the polyadenylation signal, and a poly(A) tail that is involved in the transport of the mRNA to the cytoplasm (where translation occurs) may be added.

[0045] As used herein, the "native sequence" or "natural sequence" refers to a polynucleotide or amino acid isolated from a natural source. Recombinant natural polypeptides or natural polynucleotides having the same sequence as the native form are included in the "natural sequence".

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

[0047] "Percentage of 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, wherein the portion of the polynucleotide sequence or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to a reference sequence for the optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue is present in both sequences, counting 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 of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue is present in both sequences, or the number of positions at which the nucleic acid bases or amino acid residues are 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 of sequence identity. Those skilled in the art will recognize that there are many established algorithms available for aligning two sequences.Optimal alignment of arrays 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 computerized execution 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 algorithm and the BLAST 2.0 algorithm are examples of suitable algorithms for determining the percentage of 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 from 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 satisfy a positive-valued threshold score T when aligned with words of the same length in the 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 a search to find longer HSPs that contain them. Next, the word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for pairs of matching residues; always greater than 0) and N (penalty score for mismatched residues; always less than 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of the word hits in each direction stops when the cumulative alignment score decreases by an amount X from its maximum achieved value, when the cumulative score becomes 0 or less due to the accumulation of one or more negatively 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 a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands as the default settings.For amino acid sequences, the BLASTP program uses an initial setting of word length (W) 3, expectation value (E) 10, and BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0049] The degree of the percentage of amino acid sequence identity can also be obtained by counting the number of perfect matches in the alignment by ClustalW analysis (vW1.8), dividing the number of such perfect matches by the length of the reference sequence, and using the following initial settings of ClustalW parameters to achieve slow / accurate pairwise optimal alignment, namely 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., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human). The mammal can be a domestic animal, such as a dog, cat, mouse, cow, sheep, goat, horse, or pig. These mammals can 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, the foreign nucleic acid, also referred to as exogenous nucleic acid (i.e., nucleic acid that is not part of the natural nucleic acid components of the cell), may be introduced into the cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, and conjugation, among others. Those skilled in the art will readily understand and adapt the literature that can be readily identified based on the present disclosure. The foreign nucleic acid may be administered to the subject by administering the nucleic acid or vector containing the nucleic acid described herein, for example, by intravenous administration or injection, intra-articular cartilage administration or injection, intraperitoneal administration or injection, intramuscular administration or injection, intracranial administration or injection, intraocular administration or injection, subcutaneous administration or injection, etc., and can be delivered to the subject by systemic administration to the subject.

[0052] Methods of gene therapy and methods of delivering genes to a subject, such as methods using adeno-associated virus, are described in U.S. Patent No. 6,967,018, International Publication No. 2014 / 093622, U.S. Patent Application Publication No. 2008 / 0175845, U.S. Patent Application Publication No. 2014 / 0100265, European Patent No. 2432490, European Patent No. 2352823, European Patent No. 2384200, International Publication No. 2014 / 127198, International Publication No. 2005 / 122723, International Publication No. 2008 / 137490, International Publication No. 2013 / 142114, International Publication No. 2006 / 128190, International Publication No. 2009 / 134681, European Patent No. 2341068, International Publication No. 2008 / 027084, International Publication No. 2009 / 054994, International Publication No. 2014059031, U.S. Patent No. 7,977,049, and International Publication No. 2014 / 059029, and each of these documents is hereby incorporated by reference herein in its entirety for the methods of gene delivery described therein, particularly where described in each patent or patent application.

[0053] Vector Vectors are contemplated for use with the methods and constructs described herein. The term "vector" includes nucleic acid molecules capable of transporting another nucleic acid to which it is linked. Vectors for delivering nucleic acids to cells as described herein are known to those of skill in the art and include vectors used for such purposes. In particular, specific exemplary vectors 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, which contains a DNA sequence or RNA sequence derived from a virus for packaging into a vector (e.g., retrovirus, lentivirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also include the polynucleotide carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication in the host cell 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 the host cell when introduced into the host cell and are thereby replicated with the host genome. In addition, certain vectors are capable of expressing a gene operably linked to those vectors. Such vectors are referred to herein as "expression vectors". Common expression vectors utilized in recombinant DNA technology are often in the form of plasmids.The recombinant expression vector can contain the nucleic acid of the present invention in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vector can be selected based on the host cell to be used for expression and contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. In the recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence so as to enable the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).

[0054] Examples of methods for non-viral delivery of nucleic acids or native DNA-binding proteins, native guide RNAs, or other native species include lipofection, microinjection, biolistic, virosome, liposome, immunoliposome, polycationic nucleic acid complex or lipid nucleic acid complex, naked DNA, artificial virion, and enhanced uptake of drugs by DNA. Lipofection is described in, for example, U.S. Patent 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™). Examples of cationic lipids and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those described in Felgner, International Publication Nos. 91 / 17424 and 91 / 16024. Delivery may be delivery to cells (e.g., in vitro administration or ex vivo administration) or delivery to a target tissue (e.g., in vivo administration). The term "native" includes the protein species, enzyme species, or guide RNA species themselves and does not include the nucleic acids encoding those species.

[0055] In some embodiments, the gene therapy vector used in the methods herein is a parvovirus vector, such as an animal parvovirus, and in particular, a dependovirus such as an infectious human or simian adeno-associated virus (AAV), and a vector for the introduction and / or expression of a nucleotide sequence encoding porphobilinogen deaminase in mammalian cells and its components (e.g., the animal parvovirus genome). Viruses of the Parvoviridae family are small DNA animal viruses. The Parvoviridae family can be divided into two subfamilies: the Parvovirinae subfamily that infects vertebrates and the Densovirinae subfamily that infects 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 genus Dependovirus are typically characterized by the requirement for co-infection with a helper virus such as adenovirus or herpesvirus for productive infection in cell culture. The genus Dependovirus generally includes AAVs that infect humans (e.g., serotype 1, serotype 2, serotype 3A, serotype 3B, serotype 4, serotype 5, and serotype 6) or AAVs that infect primates (e.g., serotype 1 and serotype 4), and 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). Other information about parvoviruses and other members of the Parvoviridae family is described in Kenneth 1. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed. 1996). For convenience, the present invention is further illustrated and described herein with reference to AAV. However, it should be understood that the present invention is not limited to AAV and can be equally applicable to other parvoviruses.

[0056] The genomic compositions of all known AAV serotypes are very similar. The AAV genome is a linear single-stranded DNA molecule less than about 5000 nucleotides (nt) in length. Terminal inverted repeats (ITRs) are present on both sides of the unique coding nucleotide sequences of the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, VP2, and VP3) form the capsid. The terminal 145 nucleotides (nt) are self-complementary and are configured such that an energetically stable intramolecular duplex that forms a T-shaped hairpin can be formed. These hairpin structures function as the origin of viral DNA replication and function 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 promoter and the P19 promoter, respectively, and both Rep proteins have functions in the replication of the viral genome. Splicing events in the ORF of Rep actually result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, in mammalian cells, unspliced mRNAs encoding the Rep78 protein and the Rep52 protein have been shown to be sufficient for the production of AAV vectors. Also, in insect cells, the Rep78 protein and the Rep52 protein are sufficient for the production of AAV vectors.

[0057] As used herein, "recombinant parvovirus" or "AAV vector" or "rAAV vector" refers to a vector comprising one or more polynucleotide sequences of interest, which are the genes of interest or "transgenes" to which at least one parvovirus or adeno-associated virus (AAV) inverted terminal repeat (ITR) is arranged adjacent. Such rAAV vectors can be replicated and packaged into infectious virus particles when present in insect host cells that express the AAV rep gene products and cap gene products (i.e., the AAV Rep proteins and Cap proteins). When the rAAV vector is 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 is usually referred to as a "provirus" and can be "rescued" by replication and capsid formation in the presence of the AAV packaging function and the necessary helper functions. Thus, in another aspect, the invention relates to a nucleic acid construct comprising a nucleotide sequence encoding a porphobilinogen deaminase as defined herein, which nucleic acid construct is a recombinant parvovirus vector or AAV vector and thus comprises at least one parvovirus or AAV ITR. In the nucleic acid construct, it is preferred that a parvovirus or AAV ITR is arranged adjacent to either side of the nucleotide sequence encoding porphobilinogen deaminase.

[0058] AAV can infect many mammalian cells. See, for example, 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 similar mouse cells (Jennings et al., (2001) Arthritis Res, 3:1), and the cellular tropicity of AAV varies among serotypes. See, for example, Davidson et al. (2000) Proc. Natl. Acad. Sci. USA, 97:3428-3432) which considered the differences among AAV2, AAV4, and AAV5 for mammalian CNS cell tropicity and transduction efficiency, and Goncalves, (2005) Virol J. 2(1):43 which considered approaches for modifying AAV tropicity. In some embodiments, rAAV virions having AAV1 capsid protein, AAV8 capsid protein, and AAV5 capsid protein are preferred for transduction of hepatocytes (Nathwani et al., (2007) Blood 109(4):1414-1421, Kitajima et al., (2006) Atherosclerosis 186(1):65-73), and among them, rAAV virions having AAV5 capsid protein may be most preferred.

[0059] AAV is highly prevalent within 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 is useful as a viral vector. There are many serotypes, each having tropism for different tissue types (see Zincarelli, C., et al., (2008) Mol Ther. 16(6):1073-80), which allows for preferential targeting by appropriate pseudotyping of specific tissues. Some serotypes, such as serotype 8, serotype 9, and serotype rh10, transduce mammalian bodies. See Zincarelli, C., et al., (2008) Mol Ther. 16(6):1073-80, Inagaki, K., et al., (2006) Mol Ther. 14(1):45-53, Keeler, A.M., et al., (2012) Mol Ther. 20(6):1131-8, Gray, S.J. et al., (2011) Mol Ther. 19(6):1058-69, Okada, H., et al., (2013) Mol Ther Nucleic Acids. 2:e95, and Foust, K.D., et al., (2009) Nat Biotechnol. 27(1):59-65. AAV9 has been demonstrated to cross the blood-brain barrier, a location that is difficult to reach for many viral vectors and biologics (see Foust, K.D., et al., (2009) Nat Biotechnol. 27(1):59-65, and Rahim, A.A. et al., (2011) FASEB J. 25(10):3505-18). Certain AAVs have a payload of 4.7 - 5.0 kb, including the viral inverted terminal repeats (ITRs) that are required in cis for viral packaging.See Wu, Z. et al., (2010) Mol Ther. 18(1):80-6, and Dong, J.Y. et al., (1996) Hum Gene Ther. 7(17):2101-12, both of which are incorporated herein by reference.

[0060] The VP proteins of AAV are known to determine the cell tropism of AAV virions. The VP protein coding sequences are less conserved among different AAV serotypes compared to the Rep proteins and genes. The ability of the Rep sequences and ITRs to cross-complement the corresponding sequences of other serotypes enables the production of pseudotyped rAAV particles containing the capsid proteins of one serotype (e.g., AAV5) and the Rep sequences and / or ITRs of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are part of the present invention. In this specification, pseudotyped rAAV particles may be referred to as being of the "x / y" type, where "x" indicates the origin of the ITR and "y" indicates the serotype of the capsid. For example, 2 / 5 rAAV particles have 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 at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide sequence identity and / or amino acid sequence identity to the ITR, Rep, or VP of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAvDJ, AAVrh10.XX (e.g., sequences having about 75% to about 99% nucleotide sequence identity) and can be used in place of the wild-type AAV ITR sequence, Rep sequence, or VP sequence. Preferred adenoviral vectors are modified to suppress the host response. See, for example, 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, each of which is incorporated herein by reference.

[0061] Regulatory sequences and terminators The control sequences are contemplated to be used with the gene therapy vector constructs described herein. The term "control sequences" is intended to include promoters, enhancers, internal ribosome entry sites (IRES) within the sequence, and other expression regulatory sequences (e.g., transcription termination signals such as polyadenylation signals and polyU sequences). Such control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Control sequences include sequences that constitutively express a nucleotide sequence in many types of host cells, and sequences that express this nucleotide sequence only in specific host cells (e.g., tissue-specific control sequences). Tissue-specific promoters can be used to effect expression mainly in tissues of desired purposes such as muscle, nerve, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Also, the control sequences can effect expression in a time-dependent manner, e.g., cell cycle-dependent or development stage-dependent, and the expression can be tissue-specific or cell type-specific. In some embodiments, the vector may include one or more polIII promoters (e.g., 1, 2, 3, 4, 5, or more polIII promoters), one or more polII promoters (e.g., 1, 2, 3, 4, 5, or more polII promoters), one or more polI promoters (e.g., 1, 2, 3, 4, 5, or more polI promoters), or combinations thereof. Examples of polIII promoters include, but are not limited to, the U6 promoter and the H1 promoter. Examples of polII promoters include, but are not limited to, the Rous sarcoma virus (RSV) LTR promoter (including the RSV enhancer if desired), the cytomegalovirus (CMV) promoter (including the CMV enhancer if desired).For example, see Boshart et al, (1985) Cell 41:521-530, the SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter, as well as the PolII promoters described herein. Enhancer elements such as WPRE, CMV enhancer, the R-U5’ segment in the LTR of HTLV-I (Takebe, Y. (1988) Mol. Cell. Biol. 8(1):466-472), 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 included within the term "control sequence". Those skilled in the art will understand that the design of the expression vector can depend on factors such as the choice of host cell to be transformed and the desired level of expression. The vector is introduced into the host cell to produce a transcript, protein, or peptide (e.g., a clustered regularly interspaced short palindromic repeat (CRISPR) transcript, protein, enzyme, variants thereof, fusion proteins thereof, etc.) that contains a fusion protein or fusion peptide encoded by the nucleic acids described herein.

[0062] Aspects of the methods described herein may utilize terminator sequences. A terminator sequence includes a nucleic acid sequence portion that marks the end of a gene or operon in genomic DNA during transcription. This sequence mediates transcription termination by providing a signal in the newly synthesized mRNA that causes the process of dissociating the mRNA from the transcription complex. These processes include direct interaction of the mRNA secondary structure with the complex and / or indirect activity of recruited termination factors. Dissociation of the transcription complex releases RNA polymerase and associated transcription machinery to initiate transcription of new mRNA. Terminator sequences include those known in the art and specifically described herein.

[0063] Administration, Dosage, and Treatment In various embodiments, one or more gene delivery vectors comprising a viral vector, and packaged viral particles containing the viral vector, may be in the form of a pharmaceutical or pharmaceutical composition and may be used in the manufacture of a pharmaceutical or pharmaceutical composition. The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is preferably suitable for parenteral administration. In certain embodiments, the carrier is suitable for intravenous, intra-articular, 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 a combination with sterile saline, glucose solution, or buffer solution, or other pharmaceutically acceptable sterile liquids. Alternatively, a solid carrier such as, for example, microcarrier beads may be used.

[0064] The pharmaceutical composition is usually sterile and stable under manufacturing and storage conditions. The pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other regular structure suitable for the delivery of gene therapy vectors. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it may be preferable to include in the composition isotonic agents, such as saccharides, polyalcohols such as mannitol, sorbitol, etc., or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition agents that delay absorption, such as monostearates and gelatin. The vectors of the present disclosure can be in sustained release formulations or controlled release formulations, and can be administered, for example, in compositions containing delayed release polymers, or in other carriers (including implants and microencapsulation delivery systems) that will protect the compound from rapid release. For example, biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid - polyglycolic acid copolymer (PLG) may be used.

[0065] In some embodiments, the gene therapy vector is formulated with any acceptable carrier and can be administered parenterally, such as by intravenous administration, intra - articular administration, intra - peritoneal administration, subcutaneous administration, intramuscular administration, limb perfusion, or combinations thereof. The administration can be systemic administration such that the gene delivery vector is delivered into the body of the subject. In some embodiments, the gene delivery vector can be directly administered into the target tissue. In some embodiments, the gene delivery vector can be locally administered, such as by a catheter. The route of administration can be determined by those skilled in the art, for example, considering the nature of the target tissue, the gene delivery vector, the intended therapeutic effect, and the maximum loading dose 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. "Therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic outcome, such as the treatment or amelioration of osteoarthritis, at the required dosage and for the required period. The effective amount or therapeutically effective amount of the vector may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the viral vector to elicit the desired response in the individual. The dosing regimen may be adjusted to achieve the optimal therapeutic response.

[0067] In certain embodiments, the therapeutically effective amount or prophylactically effective amount of a nucleic acid, nucleic acid construct, parvovirus virion, or pharmaceutical composition ranges from 1×10 11 ~1×10 14 genome copies (gc) / kg or from 1×10 12 ~1×10 13 genome copies (gc) / kg. It should be noted that the dosage values may vary depending on the severity of the symptoms to be alleviated. Also, the dosage may vary based on the titer of the virion used. For any particular subject, the specific dosing regimen may be adjusted over time according to the individual needs and the professional judgment of the individual administering the composition or the individual supervising the administration of the composition. The dosing ranges described herein are merely exemplary and do not limit the dosing 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 dosage range for a small animal (mouse) may be 1×10 12 genome copies (gc) / kg to 1×10 13 genome copies (gc) / kg, and for a large animal (cat or dog) and human subject, it may be 1×10 11 gc / kg to 1×10 12 gc / kg, or 1×10 11 gc / kg to 1×10 14 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. In some embodiments, several divided doses can be administered over a long period, or the dose can be relatively decreased or increased depending on the urgency of the treatment situation. In some embodiments, the gene delivery vector can be administered daily, weekly, bi - weekly, or monthly. The treatment period can be 1 week or more, 1 month or more, 2 months or more, 3 months or more, 6 months or more, or 8 months or more, or longer. In some embodiments, the treatment period can be up to 1 year or more, up to 2 years or more, up to 3 years or more, or indefinite.

[0070] In some embodiments, a therapeutically effective amount of the αKlotho protein or an active fragment thereof and a therapeutically effective amount of the sTGFβ - R2 protein or an active fragment thereof are administered to a subject for treating, for example, osteoarthritis as exhibited by articular cartilage. By the methods described herein, disorders associated with osteoarthritis or symptoms of osteoarthritis are treated or prevented, or the restoration of the structure and function of articular cartilage is improved. Remission of osteoarthritis brought about directly or via the gene therapy methods described herein by administration of the αKlotho protein or an active fragment thereof and the sTGFβ - R2 protein or an active fragment thereof is characterized by suppression of symptoms in the subject as compared to an untreated subject. In other aspects, there is provided a gene therapy method or the use of the above - mentioned nucleic acid vector for use in the treatment or prevention in a subject with osteoarthritis. According to one aspect, administration of αKlotho and sTGFβR2 coordinately suppresses or prevents the progression of osteoarthritis, for example, by down - regulating the immune response and promoting the 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 for use in the experiments described herein. Osteoarthritis is mimicked by intra-articular injection of papain, a chemical induction model that destroys cartilage microstructure and promotes proteoglycan degradation that affects 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 and thus does not impair the mechanism of cartilage regeneration that can be promoted by the treatments tested. Several osteoarthritis phenotypes associated with animal and human osteoarthritis have been reproduced by this model. For example, loss of ECM homeostasis caused by proteoglycan-degrading enzymes such as MMP13 is one of the major pathological features described for 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, Ostergaard K, Pelletier J-P, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthr Cartil. 2006 Jan;14(1):13-29). Analysis showed not only the presence of MMP13 in the ECM but also partial destruction of the cartilage structure as a result of papain treatment.

[0073] Rats (here, the osteoarthritis control group, OAC) showed grade 2 osteoarthritis as defined by the analysis parameters (see Fig. 1A). Safranin O staining showed a decrease in cartilage thickness along with discontinuous fibrous surfaces and intracellular clusters in the cartilage (see Fig. 1B). The osteoarthritis grade in these samples was further confirmed by TUNEL staining analysis and immunostaining analysis, which also showed the presence of hypertrophic chondrocytes in the joint along with the presence of cell death (see Fig. 1C), downregulation of Sox9, a chondrocyte marker (see Figs. 1F and 2A), and upregulation of Col10a and Runx2 (see Fig. 1D).

[0074] Col10a and Runx2 are well-known bone markers and, when seen in chondrocytes, are associated with the calcification of the extracellular matrix (ECM) (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). Also, the presence of proteolytic enzymes such as MMP13 in 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 of aggrecan (ACAN) and type II collagen (Col2a) (see FIGS. 1F and 2A) revealed a clear imbalance in the content of both matrix components in cartilage compared to untreated healthy knees (here the healthy control group, HC). These results indicate that in rats, osteoarthritis developed 4 weeks after papain treatment.

[0075] Example 2 Intra-articular injection using the AAV-DJ viral serotype To test the combined effects of αKlotho and sTGFβR2 on the progression and repair of osteoarthritis, these soluble factors were directly injected into the knee joint in the 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 express separate soluble factors or may express them as a fusion protein of those soluble factors.

[0076] Aspects of the present disclosure contemplate directly injecting, delivering, or administering to a patient in need of treatment the soluble factors αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof). αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof) may be administered separately by multiple formulations, for example, sequentially in succession, or may be administered together, for example, co-administered by the same formulation. Aspects of the present disclosure contemplate directly injecting, delivering, or administering to a patient in need of treatment a nucleic acid encoding the soluble factors αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof), for example, a nucleic acid within a vector such as AAV described herein as gene therapy. 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 by separate formulations, for example, sequentially in succession, or may be administered together, for example, co-administered by the same formulation.

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

[0078] Intra-articular injection of AAV-DJ-luciferase was first performed to test the safety of this method. According to the luciferase measurements, intra-articular injection enabled limiting AAV infection to the knee joint without invading the bloodstream with the injection and avoiding effects on other tissues (see Figure 3A). This can help avoid causing side effects. First, an in vitro analysis using AAD-DJ-GFP in vitro was performed to test the infectivity efficacy of the AAV-DJ serotype. From the results analyzed by flow cytometry, a significantly higher efficiency was shown in synovial mesenchymal cells compared to chondrocytes, although both populations were transduced (see Figures 3B and 3C). The high infectivity efficacy of mesenchymal stem cells can help avoid harmful cell effects on chondrocytes as a result of direct AAV infection (see 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 aspect, both αKlotho and sTGFβR2 are released by adjacent mesenchymal cells that are localized in the joint and will exert their effects throughout the joint.

[0079] Example 3 αKlotho and sTGFβR2 improve the clinical score of osteoarthritis rats by preventing and reversing the osteoarthritis phenotype To test the possibility that αKlotho and sTGFβR2 are effective in cartilage repair, early osteoarthritis was induced in papain-treated rats for 4 weeks, and then those rats were treated by intra-articular injection of either AAV-DJ-GFP (sham group) or AAV-DJ-αKlotho and AV-DJ-sTGFβR2 (hereafter the KT group) (a schematic diagram can be seen in Fig. 2B).

[0080] Rats injected with AAV-DJ-GFP showed greater cartilage deterioration after 6 weeks. As demonstrated by safranin O staining, Col2a staining, and ACAN staining, due to significant downregulation of ECM components in the remaining fragments (see Fig. 4A, Fig. 4B, and Fig. 2A), not only erosion and obvious disappearance of cartilage structure but also matrix calcification were shown. Immunohistological analysis showed a significant decrease in the number of Sox9+ cells (see Fig. 4B and Fig. 2A), but apoptotic cells (see Fig. 4C), hypertrophy markers (see Fig. 4D), and MMP13 (see Fig. 4E) were still seen in the remaining cartilage segments. As a result, the cartilage thickness decreased dramatically (see Fig. 2C) (i.e., thinning of cartilage as a symptom of osteoarthritis), and the injury was classified as grade 4 by OARSI score analysis (see Fig. 4F), indicating a clear progression to the pathological condition of osteoarthritis.

[0081] Rats treated with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2 showed a significant improvement in phenotype after 6 weeks. Separate AAV-DJ viruses were produced using separate vectors. A total of 2.5×10 1250% sTGFβR2 and 50% αKlotho of GC were injected into the joint. Intra-articular injection of AAV expressing αKlotho and sTGFβR2 not only avoided the release of MMP13 into the ECM, but also promoted the maintenance of cartilage thickness. Compared with the OAC group, safranin O staining (see Fig. 4A) showed the recovery of cartilage thickness (see Fig. 2C) and structure. First, the outermost layer in which cells are arranged in horizontal clusters parallel to the surface of the joint and composed of string-like, paired, and single cells. Second, the middle and deep layers containing two or more layers of chondrocytes arranged in vertical columns. Positive staining for Col2a and ACAN supports the functional recovery of chondrocytes accompanied by the regeneration of ECM components in the joint (see Figs. 1B and 2A). The suppression of ECM degradation by αKlotho supports the enhancement of Col2a staining, ACAN staining, and safranin O staining after KT treatment. This was further evaluated in the analysis of tissue sections. This data not only shows the complete absence of apoptotic cells in the joint (see Fig. 4C), but also shows the restoration of the distribution of hypertrophic markers. In contrast to the OAC group and the sham group, in the KT-treated joints, most of the positive cells for Col10a and Runx2 are localized at the lower layer level of the cartilage layer corresponding to the regular hypertrophic layer of cartilage, similar to the HC group (see Fig. 4D). Also, the presence of the protease MMP13 was not detected in the ECM of the HC and KT-treated knees (see Fig. 4E). Based on the significant improvement of the joint, the OARSI classification shows that the rats treated with αKlotho and sTGFβR2 recovered from grade 2 to grade 1 osteoarthritis, while the rats treated with AAV-DJ-GFP progressed to grade 4 osteoarthritis (see Fig. 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 receptor / ALK5 receptor ratio during aging or upon long-term exposure to TGF-β1, indicating the importance of maintaining the balanced TGFβ pathway. Thus, the high affinity of the TGFβR2 receptor for TGFβ1 and TGFβ3 suppresses chondrocyte hypertrophy and downregulates hypertrophy markers after KT treatment.

[0083] According to one aspect, the use of both αKlotho and sTGFβR2 contributes to the recovery of the ECM, for example, by maintaining the balance between the anabolic and catabolic pathways. The TGFβ1 pathway is thought to be a repair mediator by stimulating chondrocyte proliferation. According to one aspect, using sTGFβR2 to capture TGFβ1 suppresses the catabolic pathway but enhances its anabolic action.

[0084] Example 4 αKlotho and sTGFβR2 improve the inflammatory response characteristic of the osteoarthritis phenotype Although osteoarthritis was initially classified as non-inflammatory 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 significant loss of articular cartilage and joint space narrowing in osteoarthritis (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 examine some of the underlying mechanisms of the effects of αKlotho and sTGFβR2 on osteoarthritis, cartilage tissues were isolated from all groups for RNA sequencing analysis. RNA sequencing analysis revealed differentially expressed (DE) genes in the KT group when compared to the OAC group and sham group. Specifically, 489 genes were differentially expressed in KT compared to sham, and 156 of these genes showed a similar expression pattern between KT and HC. Gene ontology (GO) analysis showed that among these differentially expressed genes, the gene groups involved in inflammatory and immune responses showed the most dramatic effects upon KT treatment (see FIGS. 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). Pro-inflammatory cytokines associated with nuclear factor (NF)-κB and interleukin-1 (IL)-1β have been described as promoting 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-alpha is mediated by MAP kinases, AP-1, and NF-kappaB transcription factors in articular chondrocytes. Exp Cell Res. 2003 Aug 1;288(1):208-17).Therefore, when comparing the OAC group and the sham group with KT, the data showed downregulation of (1) interleukin-related genes such as Il1rn (see FIGS. 5D and 7), (2) Tnf-related / NF-κB-dependent genes such as Tnfaip2 (see FIGS. 5D and 7), (3) interferon-related genes such as the Ifit gene (see FIGS. 5C and 5D), and (4) cytokines or chemokines such as Ccl6 (see FIGS. 5C and 5D) (see 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] Therefore, data demonstrate that chondrocytes already show upregulation of pro-inflammatory cytokines and immune response-related factors 4 weeks after papain treatment. Treatment with αKlotho and sTGFβR2 not only downregulates the expression of some of these already expressed genes, but also avoids subsequent upregulation of other immune response factors, thereby demonstrating the role of TGFβ in inflammation during osteoarthritis. TGFβ induces synovial lining cells to produce inflammatory factors, and these inflammatory factors can further stimulate the hypertrophy of articular chondrocytes (see Scanzello CR, Goldring SR. The role of synovitis in osteoarthritis pathogenesis. Bone. 2012 Aug;51(2):249-57). Blockade of TGFβ signaling significantly attenuates synovial hyperplasia involved in the pathogenesis of osteoarthritis (see 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 involved in the cellular inflammatory response.Various studies have shown how treatment with recombinant Klotho can reduce cytokine levels involved in kidney and heart diseases (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 aspect, the synergistic activity of both αKlotho and sTGFβR2 reduced the osteoarthritis-related inflammatory response.

[0088] During the inflammatory reaction that occurs in osteoarthritis, nitric oxide (NO) produced by Nos2 has a destructive effect that causes 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, together 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, aspects of the present disclosure relate to the use of αKlotho (or an active fragment thereof) and sTGFβR2 (or an active fragment thereof) as soluble factors, either directly or via gene therapy, to avoid or reduce subsequent destructive processes induced by an inflammatory response. According to one aspect (see FIGS. 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 avoided cartilage degradation by reducing IL-1β-induced NO production through a decrease in the mRNA levels of Il1rn and Nos2 in chondrocytes. According to one aspect, αKlotho reduces oxidative stress and downregulates apoptosis during KT treatment (see 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. Geriatr Gerontol Int. 2011 Oct;11(4):510-6).

[0090] Example 5 αKlotho and sTGFβR2 promote the expression of human chondrocyte markers in vitro To evaluate the potential of KT treatment to be effective in human cartilage, the in vitro effects of αKlotho and sTGFβR2 were tested using primary human articular chondrocytes. The phenotype of articular cartilage is characterized by the expression of cartilage-specific extracellular matrix components, mainly Col2a, and the expression of Sox9, a cartilage-specific transcription factor. The expression of Sox9 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). The maintenance of this differentiated phenotype in vitro depends greatly on the 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 dedifferentiation or hypertrophy of chondrocytes (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, using two separate vectors described herein, the effects of both αKlotho and sTGFβR2 factors on the phenotypic characteristics of human hyaline chondrocytes under monolayer culture conditions were tested. First, to mimic the in vivo model, the virus was infected into mesenchymal cells to approach chondrocytes. For this purpose, a co-culture experiment as described herein was designed in which human fibroblasts are efficiently infected with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2 (KT), or infected with AAV-DJ-GFP as a control (see Figure 6A). From these results, it was shown that mesenchymal cells transduced with KT showed a high proportion of chondrocytes expressing Sox9 and Col2a, which are chondrocyte-specific markers essential for cell identity and ECM formation, respectively (see Figure 6B). An increase in the number of cells progressing through the cell cycle was also observed in this culture (see Figures 6B and 6C), which supports the effect of αKlotho on cell proliferation. Therefore, a method for regrowing cartilage is provided by administering αKlotho or its active fragment and sTGFβR2 or its active fragment, or by administering the gene in a vector for expression, for example, treatment with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2.

[0092] Also, human articular chondrocytes were treated in vitro for 10 days with αKlotho and sTGFβR2. These results showed similar clear improvements demonstrated by induction of the expression of Sox9 protein and Col2a protein and enhancement of cell proliferation (see Figure 6D). Therefore, a method for treating human hyaline cartilage is provided by administering αKlotho or its active fragment and sTGFβR2 or its active fragment, or by administering the gene in a vector for expression, for example, treatment with AAV-DJ-αKlotho and AAV-DJ-sTGFβR2. Therefore, αKlotho and sTGFβR2 are administered to maintain the cartilage phenotype in humans.

[0093] Example 6 Methods and Reagents Isolation and Culture of Cells Human articular cartilage was harvested from healthy donors according to informed consent for medical research, and rat articular cartilage was removed from the femoral condyles and tibial condyles of healthy rats under aseptic conditions. Isolation and culture of chondrocytes 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 joint capsule connective tissue. Briefly, the 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 the stromal vascular cell population was isolated by centrifugation and maintained in basal medium.

[0095] Cloning and Production of AAV AAV plasmids were constructed according to the following standard cloning techniques. The following primers were used to PCR - amplify α - Klotho and sTGFβR2.

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

[0096] With minor modifications, AAV was prepared using 293AAV cells (Cell Biolabs, Inc.) as described (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 the virus was purified by CsCl density gradient. The virus titer was determined by qPCR using the following primers.

Chem.

[0097] Osteoarthritis injury model Experimental induction of osteoarthritis was performed 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 (on days 1, 4, and 7). All animal experiments were conducted according to protocols approved by the IACUC and the Salk Institute's Division of Animal Resources.

[0098] Design of animal experiments Twenty 250 g female Sprague-Dawley (SD) rats were divided into four groups: a healthy control group (HS) of 8 animals per group, an osteoarthritis control group (OAC), an osteoarthritis group treated with αKlotho and sTGFβR2 for 4 weeks (KT), and a 4-week osteoarthritis sham group (sham). The 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 grade of osteoarthritis reached at this time. The other two 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 in 50 μl of PBS per knee 12 GC was injected. These last two groups were sacrificed 6 weeks after virus injection. The joints of both knees were collected from each rat, one knee for histological analysis and the other knee for RNA isolation. All animal experiments were conducted according to protocols approved by the IACUC and the Salk Institute's Division of Animal Resources.

[0100] RNA extraction The cartilage surface was washed with physiological saline and then dissected from the articular surface using a razor blade. Care was taken to avoid contamination with blood, bone, or synovium. The tissue was cut into small pieces, immersed in TRIzol (Ambion), immediately snap-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 electrophoresed on a TapeStation automated electrophoresis analysis system (2200 TapeStation) according to the manufacturer's instructions. The RNA concentration was determined using a Qubit fluorometer 2.0.

[0101] Histology and immunofluorescence 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), the entire knee joint was prepared for histology. Samples were sectioned 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 Kawamot’s film method (2012). Methods Mol Biol. 2014;1130:149-64). Next, the sections were stained using various methods. Safranin O / fast green staining was performed according to standard techniques. The grade of pathological changes in articular cartilage was determined using the Osteoarthritis Research Society International (OARSI) scoring system according to the above methodology (23). That is, normal and healthy cartilage was given a grade of 0, grade 1 was applied when the cartilage surface was intact but included worn areas, hypertrophy, and cell clusters, grade 1.5 was grade 1 including cell death, grade 2 cartilage showed an intermittent fibrous surface, grade 2.5 consisted of grade 2 including matrix loss indicated by less than 1 / 3 safranin O staining, grade 3 was determined when cracks appeared up to the middle layer and less than 2 / 3 of the cartilage was stained with safranin O, grade 3.5 showed deeper cracks into the middle layer, grade 4 indicated matrix loss due to exfoliation of the outermost layer, grade 4.5 showed a depression into the middle layer, grade 5 cartilage showed completely eroded non-calcified cartilage, grade 5.5 showed growth of hypertrophic cartilage after erosion, and grade 6, a higher grade, showed more severe cartilage damage when deformation of the articular eminence appeared.

[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). Images were taken using a slide scanning microscope (Olympus VS-120 Virtual Slide Scanning Microscope).

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

[0105] Antibodies Collagen type II (NeoMarkers) diluted 1:100 (volume / volume), Runx2 (Santa Cruz Biotechnology) diluted 1:100 (volume / volume), MMP13 (Abcam) diluted 1:100 (volume / volume), MMP3 (Abcam) diluted 1:100 (volume / volume), Sox9 (Abcam) diluted 1:100 (volume / volume), collagen X (Abcam) diluted 1:50 (volume / volume), and Ki67 (BioLegend) diluted 1:100 (volume / volume). Antigen activation was performed by heat-mediated treatment with 5% hyaluronidase in acetate buffer at 37°C for 1 hour. 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 300 g long - Evans rats were used to test intra - articular injection of AAV - DJ, namely AAV - DJ - Luc or AAV - DJ (empty vector, negative control). Two weeks after injection, luciferase was detected using an IVIS Kinetic 2200 (Caliper Life Sciences). 50 mg / kg of D - luciferin (Biosynth) was injected intra - articularly and intra - peritoneally. Images were acquired 10 minutes after the injection of D - luciferin.

[0107] RNA Sequencing and Data Analysis Reads were mapped to the 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)] with default parameters. Only uniquely mapped reads were used for downstream analysis. Gene expression levels were calculated by summing reads mapped across all exons of RefSeq genes using HOMER [v4.8 (Homer Software and data download [Internet]. Available from the World Wide Web 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 from the World Wide Web ncbi.nlm.nih.gov / pmc / articles / PMC4302049 / )] with cut - offs logFC (log fold change)>0.5 and FDR (false discovery rate)<0.05.

[0108] Enrichment tests 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), where the genes of interest were used as input signals and the entire rn6 genome was used as the background. Only biological process terms with Benjamini-Hochberg FDR < 0.01 were used. Data can be accessed with the GEO accession number GSE118559.

[0109] Unless specifically mentioned, all statistical analyses of RNA sequences were performed in the R environment [v3.4.3, (R: A Language and Environment for Statistical Computing [Internet]. Available from World Wide Web gbif.org / tool / 81287 / r-a-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 from World Wide Web 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 Visualisations Using the Grammar of Graphics [Internet]. Available from World Wide Web CRAN.R-project.org / package=ggplot2).

[0110] in vitro experiments Co-culture experiment: The trans-in vitro effects of sTGFβR2 and αKlotho on human chondrocytes were evaluated using Corning® Transwell® polyester membrane cell culture inserts with a pore size of 4 μm (Sigma) in a 24-well plate. Briefly, 3×10 12 p100 plates of human fibroblasts were transduced with AAV-DJ-αKlotho and AAV-DJ-TGFβR2. Two days after transduction, cells were re-seeded into the upper well of the co-culture chamber, and chondrocytes were seeded into the lower well of this chamber on the same day. The cells in the chamber were cultured for 10 days at 37 °C and 5% CO2.

[0111] Soluble factor experiment: Chondrocytes were seeded onto plates at a 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. The cells were cultured at 37 °C and 5% CO2, and the medium containing the above factors or BSA was replaced every 3 days.

[0112] Immunofluorescence of cell cultures Before fixation, cells were cultured with EdU for 2 hours according to a standard protocol. Next, the cells were fixed using 4% PFA. The cells were permeabilized with 0.1% Triton X-100 in PBS for 20 minutes at room temperature (RT). After washing with PBS, the cells were blocked with 1% BSA in PBS for 1 hour and incubated overnight at 4 °C with primary antibodies (type II collagen diluted 1:150 and Sox9 diluted 1:300). The secondary antibody was 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 obtained using a Zeiss LSM 880 Rear Port Laser Scanning Confocal.

[0113] Flow cytometry analysis After filter filtration (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 presented as mean ± standard error (s.e.). Welch's correction (in the absence of the premise of equal s.d. for each group) was performed, and statistical significance ( * P value) was determined by an unpaired Student's two-tailed t-test. All analyses were performed using Prism7 software from GraphPad (San Diego, California, USA). Statistical significance was defined as P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0115] Example 7 Gene therapy for osteoarthritis using systemic injection of sTGFbR2 + αKlotho The lesions of osteoarthritis (OA) are induced by intra-articular injection of papain as described herein and consist of a complete cartilage surface mainly with surface fibrosis, chondrocyte cell death / loss and proliferation, edema, and / or loss of the outermost proteoglycan matrix. Occasionally, deeper fibrosis, wear, and cracks extending to the intermediate layer were observed. Also, surface fibrosis was observed along the surface of the meniscus. Fibrosis was characterized by small cracks and discontinuities in the cartilage matrix at the outermost surface or meniscus surface. Chondrocyte cell death / loss was characterized by the absence of chondrocytes or "ghost" chondrocytes present in the outermost and intermediate layers of the cartilage. Chondrocyte proliferation was characterized by an increase in the number of chondrocytes, often in a disorganized manner, within the outermost and intermediate layers of the cartilage. Edema was characterized by an increase in clear fluid around chondrocytes in the outermost and intermediate layers of the cartilage. Loss of the proteoglycan matrix was characterized by a decrease or loss of cationic staining (red, safranin O), and the loss of the matrix may be present closest to surviving chondrocytes or in areas where chondrocytes have been lost and was usually accompanied by other lesions as described above. Wear was characterized by a localized loss of the outermost layer that roughened the surface. Cracks were characterized by a longitudinal separation of the matrix extending to the intermediate layer cartilage. To evaluate a gene therapy for rat knee osteoarthritis, a viral vector containing nucleic acids encoding sTGFbR2 and FGF21 was injected systemically into rats.

[0116] As shown in Figure 8, the OA grade was determined based on the depth of the most severe lesions found in the samples (outermost, intermediate, deep, or bone infiltration). The control group (Group 2) and the sTGFbR2+FGF21 treatment group (Group 4) two months after papain treatment showed the highest grades, and the control group (Group 1) one month after papain treatment showed the lowest grade. A slight decrease in grade was observed in the sTGFbR2+αKlotho treatment group (Group 3) compared to the control group (Group 2) two months after papain treatment.

[0117] As shown in Fig. 9, the OA stage was determined based on the entire range of cartilage affected by OA lesions. Among the groups, the trend was similar to that observed for OA grade, but the lowest average score for OA stage was observed in the sTGFbR2+αKlotho treatment group (Group 3).

[0118] As shown in Fig. 10, the OA score was determined by multiplying the grade and stage for all OA values. Among the groups, the trend was similar to that observed for OA stage. The group 2 months after papain treatment (Group 2) and the sTGFbR2+FGF21 treatment group (Group 4) showed equivalent highest scores. The group 1 month after papain treatment (Group 1) had a lower score compared to the group 2 months after papain treatment (Group 2). The sTGFbR2+αKlotho treatment group (Group 3) showed the lowest score and a decrease in lesion severity compared to the two control groups (Group 1 and Group 2).

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

[0120] Example 8 Comparison of treatments using combinations of αKlotho and sTGFbR2 independently First, by using an osteoarthritis in vitro model that uses high concentrations of TGFβ1, the combined effects of αKlotho ("K") and sTGFβR2 ("T"), identified as ("KT"), were analyzed. From the results analyzed by qPCR, it was shown how the combination of both soluble factors synergistically supported the inhibition of hypertrophy markers and the inhibition of ECM proteases when compared to treatment with single factors (Figure 12A, KT versus K and T). Thus, chondrocytes treated with both factors also showed higher protein expression of ACAN than αKLOTHO or sTGFβR2 (Figure 12B). Figure 12C illustrates the injection timeline.

[0121] Example 9 Embodiment An embodiment of the present disclosure is a method of treating osteoarthritis in a subject in need thereof, comprising 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. 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 a 2A sequence.

[0122] Embodiments of the present disclosure are methods for treating osteoarthritis in a subject in need of treatment, comprising 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 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 certain aspects, 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 certain aspects, 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. According to certain aspects, each of the first regulatory sequence and the second regulatory sequence comprises a promoter. According to certain aspects, the promoter is a constitutive promoter or an inducible promoter. According to certain aspects, each of the first regulatory sequence and the second regulatory sequence comprises a cell-specific promoter or a tissue-specific promoter. According to certain aspects, each of the first regulatory sequence and the second regulatory sequence comprises a liver-specific promoter. According to certain aspects, the regulatory sequence comprises a promoter selected from the group consisting of the hEf1α promoter, the shEf1α promoter (or truncated hEf1α promoter), the CAG promoter (e.g., cytomegalovirus, chicken β-actin intron, rabbit β-globin gene splice acceptor), the CMV promoter, the hAAT promoter, the thyroxine-binding globulin promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, the liver control region (HCR)-ApoCII hybrid promoter, the CASI promoter, the HCR-hAAT hybrid promoter, the hAAT promoter combined with the mouse albumin gene enhancer (Ealb) element, and the apolipoprotein E promoter. According to certain aspects, the first nucleic acid sequence is operably linked to a 3' untranslated region for RNA stability and expression in mammalian cells.According to one aspect, the 3' untranslated region includes a sequence selected from the group consisting of a WPRE sequence, a WPRE3 sequence, an SV40 late polyadenylation signal (e.g., a shortened form), an HBG polyadenylation signal, a rabbit β-globin polyadenylation signal, a bovine bgpA, an ETC polyadenylation signal, and any combination thereof. According to one aspect, the first viral vector and / or the second viral vector is an adeno-associated virus (AAV) vector. According to one aspect, the AAV vector is AAV-DJ. According to one aspect, the AAV vector is derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, and AAVrh10.XX viral vectors. According to one aspect, the sTGFβ-R2 protein is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine sTGFβ-R2 proteins. According to one aspect, the sTGFβ-R2 protein is a human sTGFβ-R2 protein. According to one aspect, the sTGFβ-R2 protein is a canine sTGFβ-R2 protein. According to one aspect, the sTGFβ-R2 protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 3. According to one aspect, the αKlotho protein is selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine, and porcine αKlotho proteins. According to one aspect, the αKlotho protein is a human αKlotho protein. According to one aspect, the αKlotho protein is a canine αKlotho protein. According to one aspect, the αKlotho protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 1. According to one aspect, the sTGFβ-R2 protein and / or the αKlotho protein is an Fc fusion protein comprising an Ig Fc domain.According to one aspect, the Ig Fc domain is selected from the group consisting of the Fc of human, dog, cat, cow, sheep, goat, horse, mouse, and pig, or the subtypes of the Fc including IgG1, IgG2a, IgG2b, IgG3, and IgG4. According to one aspect, the Ig Fc domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. According to one aspect, the sTGFβ-R2 protein and / or the αKlotho protein are expressed and distributed systemically. According to one aspect, the first viral vector and / or the second viral vector are administered by intravenous injection. According to one aspect, 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 aspect, the first viral vector and / or the second viral vector infect mesenchymal cells at the site of osteoarthritis. According to one aspect, treating osteoarthritis in the subject comprises suppressing the progression of osteoarthritis in the subject as compared to a control subject. According to one aspect, treating osteoarthritis in the subject comprises an increase, regeneration, or regrowth of cartilage at the site of osteoarthritis in the subject as compared to a control subject. According to one aspect, treating osteoarthritis in the subject comprises suppressing inflammation at the site of osteoarthritis in the subject as compared to a control subject. According to one aspect, the subject is a mammal. According to one aspect, the mammal is a human. According to one aspect, the mammal is a dog.

[0123] Embodiments of the present disclosure are methods for treating osteoarthritis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination of an αKlotho protein or an active fragment thereof and an sTGFβ-R2 protein or an active fragment thereof, thereby treating osteoarthritis in the subject. According to one aspect, 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 aspect, the αKlotho protein and / or the sTGFβ-R2 protein is administered by intravenous injection. According to one aspect, the αKlotho protein and / or the sTGFβ-R2 protein is administered by intra-articular injection into the cartilage at the site of osteoarthritis. According to one aspect, treating osteoarthritis in the subject comprises suppressing the progression of osteoarthritis in the subject as compared to a control subject. According to one aspect, treating osteoarthritis in the subject comprises increasing, regenerating, or regrowing cartilage at the site of the osteoarthritis in the subject as compared to a control subject. According to one aspect, treating osteoarthritis in the subject comprises suppressing inflammation at the site of osteoarthritis in the subject as compared to a control subject.

[0124] Embodiments of the present disclosure are methods for treating osteoarthritis in a subject in need thereof, 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 an sTGFβ-R2 protein or an active fragment thereof, thereby treating osteoarthritis in the subject. According to certain aspects, the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element. According to certain aspects, the polycistronic element is an IRES or a 2A sequence. According to certain aspects, the nucleic acid molecule is administered by intravenous injection. According to certain aspects, the nucleic acid molecule is administered by intra-articular injection into cartilage at the site of osteoarthritis. According to certain aspects, treating osteoarthritis in the subject comprises suppressing the progression of osteoarthritis in the subject as 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 as compared to a control subject. According to certain aspects, treating osteoarthritis in the subject comprises suppressing inflammation at the site of osteoarthritis in the subject as compared to a control subject. According to certain aspects, the nucleic acid molecule comprises DNA, RNA, or a combination thereof. According to certain aspects, the subject is a mammal. According to certain aspects, the mammal is a human. According to certain aspects, 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. According to certain aspects, the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element. According to certain aspects, the polycistronic element is an IRES or a 2A sequence. According to certain aspects, 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. According to certain aspects, the first promoter and the second promoter are cell-specific or tissue-specific. According to certain aspects, the first promoter and the second promoter are constitutive or inducible. According to certain aspects, 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 certain aspects, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins. According to certain aspects, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins. According to certain aspects, the αKlotho protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. According to certain aspects, the sTGFβ-R2 protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0126] Embodiments of the present disclosure relate to a pharmaceutical formulation comprising the above-described vector and a pharmaceutically acceptable excipient.

[0127] Embodiments of the present disclosure are methods for treating osteoarthritis in a mammal in need thereof, comprising administering a therapeutically effective amount of a combination of an αKlotho protein or an active fragment thereof and an sTGFβ-R2 protein or an active fragment thereof to the mammal at a site exhibiting osteoarthritis in the mammal, wherein the progression of the osteoarthritis is suppressed as compared to an untreated state, cartilage at the site of the osteoarthritis increases, regenerates, or regrows as compared to an untreated state, or inflammation is suppressed as compared to an untreated state. According to one aspect, the mammal is a dog or a human. According to one aspect, 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 aspect, the αKlotho protein or an active fragment thereof is administered by intra-articular cartilage injection as a soluble protein, and the sTGFβ-R2 protein or an active fragment thereof is administered by intra-articular cartilage injection as a soluble protein. According to one aspect, 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, 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 aspect, 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, 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 aspect, 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, 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 aspect, 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, 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 aspect, the vector is a recombinant virus. According to one aspect, the vector is a parvovirus. According to one aspect, the vector is an AAV vector. According to one aspect, the AAV vector is AAV-DJ. According to one aspect, the vector is an AAV vector classified into a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrh10.XX, or a combination thereof. According to one aspect, the vector infects mesenchymal cells at the site of the osteoarthritis. According to one aspect, the first vector and the second vector are recombinant viruses. According to one aspect, the first vector and the second vector are parvoviruses. According to one aspect, the first vector and the second vector are AAV vectors. According to one aspect, 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 a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, or AAVrh10.XX, or a combination thereof.According to one aspect, the first vector and the second vector infect mesenchymal cells at the site of osteoarthritis. According to one aspect, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins. According to 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 aspect, the αKlotho protein and the sTGFβ-R2 protein are selected from the group consisting of proteins of humans, dogs, cats, cows, sheep, goats, horses, mice, and pigs. According to one aspect, the αKlotho protein has at least 90% sequence identity to the amino acid sequence of the αKlotho protein corresponding to SEQ ID NO: 1. According to one aspect, the sTGFβ-R2 protein has at least 90% 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 β receptor II (sTGFβ-R2) protein or an active fragment thereof. According to certain aspects, 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. According to certain aspects, the first promoter and the second promoter are cell-specific or tissue-specific. According to certain aspects, the first promoter and the second promoter are constitutive or inducible. According to certain aspects, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are human proteins. According to certain aspects, the αKlotho protein or an active fragment thereof and the sTGFβ-R2 protein or an active fragment thereof are canine proteins. According to certain aspects, 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 certain aspects, the αKlotho protein has at least 90% sequence identity to the amino acid sequence of the αKlotho protein corresponding to SEQ ID NO: 1. According to certain aspects, the sTGFβ-R2 protein has at least 90% sequence identity to the amino acid sequence of the sTGFβ-R2 protein corresponding to SEQ ID NO: 3.

[0129] Embodiments of the present disclosure relate to a pharmaceutical formulation comprising the above-described vector in a pharmaceutically acceptable excipient.

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

[0131] Although various specific embodiments have been illustrated and described, it should be understood that various changes can be made without departing from the spirit and scope of the present invention.

[0132] Sequence Listing [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

Claims

1. A first viral vector comprising a first nucleic acid sequence encoding a soluble transforming growth factor β receptor II (sTGFβ-R2) protein or an active fragment thereof and a second nucleic acid sequence encoding an αKlotho protein or an active fragment thereof, or A first viral vector comprising a first nucleic acid sequence encoding a soluble transforming growth factor β receptor II (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, comprising A pharmaceutical composition comprising a viral vector, which is used for treating osteoarthritis in a subject in need of treatment.

2. The pharmaceutical composition according to claim 1, wherein the first viral vector comprises the first nucleic acid sequence and the second nucleic acid sequence, and the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element selected from the group consisting of an IRES and a 2A sequence.

3. The first viral vector and / or the second viral vector is a recombinant virus, a parvovirus, an adeno-associated virus (AAV) vector, and / or an AAV vector derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2.5, AAVrh10.XX, and AAV-DJ, The pharmaceutical composition according to claim 1.

4. The pharmaceutical composition according to claim 1, wherein the sTGFβ-R2 protein or an active fragment thereof and / or the αKlotho protein or an active fragment thereof are expressed and distributed systemically in the subject.

5. An αKlotho protein or an active fragment thereof, and a soluble transforming growth factor β receptor II (sTGFβ-R2) protein or an active fragment thereof comprising A pharmaceutical composition used for treating osteoarthritis in a subject in need of treatment.

6. The pharmaceutical composition according to claim 5, wherein the αKlotho protein or an active fragment thereof is administered to the subject as a soluble protein, and the sTGFβ-R2 protein or an active fragment thereof is administered to the subject as a soluble protein.

7. Intravenous injection, or Intra-articular injection into cartilage at the site of osteoarthritis administered to the subject by The pharmaceutical composition according to claim 1 or claim 5.

8. The pharmaceutical composition according to claim 7, which is dependent on claim 1, wherein the first viral vector and / or the second viral vector infects mesenchymal cells at the site of osteoarthritis.

9. The treatment of osteoarthritis in a subject is inhibition of the progression of osteoarthritis in the subject compared to a control subject, increase, regeneration, or regrowth of cartilage at the site of osteoarthritis in the subject compared to a control subject, and / or inhibition of inflammation at the site of osteoarthritis in the subject compared to a control subject The pharmaceutical composition according to claim 1 or claim 5, comprising

10. The pharmaceutical composition according to claim 9, wherein the subject is a mammal selected from the group consisting of humans and dogs.

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

12. The vector according to claim 11, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element selected from the group consisting of IRES and 2A sequences.

13. The first promoter is operably linked to the first nucleic acid sequence for the expression of sTGFβ-R2 protein or an active fragment thereof in mammalian cells, The second promoter is operably linked to the second nucleic acid sequence for the expression of αKlotho protein or an active fragment thereof in mammalian cells, The vector according to claim 11.

14. A pharmaceutical composition comprising the vector according to any one of claims 11 to 13 and a pharmaceutically acceptable inactive ingredient (excipient).

15. The first nucleic acid sequence is operably linked to a first regulatory sequence that causes the expression of the sTGFβ-R2 protein or an active fragment thereof, and / or The second nucleic acid sequence is operably linked to a second regulatory sequence that causes the expression of the αKlotho protein or an active fragment thereof, The pharmaceutical composition according to any one of claims 1 to 4 or the vector according to any one of claims 11 to 13.

16. Each of the first regulatory sequence and the second regulatory sequence includes a promoter. Each of the first regulatory sequence and the second regulatory sequence includes a constitutive promoter or an inducible promoter. Each of the first regulatory sequence and the second regulatory sequence includes a cell-specific promoter or a tissue-specific promoter. Each of the first regulatory sequence and the second regulatory sequence includes a liver-specific promoter, and / or Each of the first regulatory sequence and the second regulatory sequence includes a promoter selected from the group consisting of the hEf1α promoter, the shEf1α promoter (or truncated hEf1α promoter), the CAG promoter (e.g., cytomegalovirus, chicken β-actin intron, splice acceptor of rabbit β-globin gene), the CMV promoter, the hAAT promoter, the thyroxine-binding globulin promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, the liver control region (HCR)-ApoCII hybrid promoter, the CASI promoter, the HCR-hAAT hybrid promoter, the hAAT promoter combined with the mouse albumin gene enhancer (Ealb) element, and the apolipoprotein E promoter. The pharmaceutical composition or vector according to claim 15.

17. The first nucleic acid sequence is operably linked to a 3' untranslated region for RNA stability and expression in mammalian cells, and / or The first nucleic acid sequence is operably linked to a 3' untranslated region comprising a sequence selected from the group consisting of the WPRE sequence, the WPRE3 sequence, the SV40 late polyadenylation signal (e.g., truncated), the HBG polyadenylation signal, the rabbit β-globin polyadenylation signal, the bovine bgpA, the ETC polyadenylation signal, and any combination thereof. The pharmaceutical composition according to any one of claims 1 to 4 or the vector according to any one of claims 11 to 13.

18. The first nucleic acid sequence and / or the second nucleic acid sequence includes DNA, RNA, or a combination thereof. The pharmaceutical composition according to any one of claims 1 to 4 or the vector according to any one of claims 11 to 13.

19. The sTGFβ-R2 protein or its active fragment and / or the αKlotho protein or its active fragment is selected from the group consisting of proteins or their active fragments of human, dog, cat, bovine, sheep, goat, horse, mouse, and pig. Here, The sTGFβ-R2 protein or its active fragment may include an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

3. The αKlotho protein or its active fragment may include an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1. The pharmaceutical composition according to any one of claims 1 to 6 or the vector according to any one of claims 11 to 13.

20. The sTGFβ-R2 protein or its active fragment and / or the αKlotho protein or its active fragment is an Fc fusion protein containing an Ig Fc domain. The pharmaceutical composition according to any one of claims 1 to 6 or the vector according to any one of claims 11 to 13.

21. The Ig Fc domain is selected from the group consisting of Fc of human, dog, cat, bovine, sheep, goat, horse, mouse, and pig, or subtypes of the Fc including IgG1, IgG2a, IgG2b, IgG3, and IgG4, and / or The Ig Fc domain includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7. The pharmaceutical composition or vector according to claim 20.

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