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  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/495—Transforming growth factor [TGF]
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  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12N2750/14011—Parvoviridae
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

• articular cartilage is a tissue that undergoes substantial changes in the matrix structure, molecular composition, metabolic activity, and mechanical properties (see Rahmati M,
• osteoarthritis is the most prevalent musculoskeletal disorder among the elderly and is the leading cause of disability in the US due to pain associated with the disease (Zhang Y, Jordan JM. Epidemiology of Osteoarthritis. Clin Geriatr Med. 2010 Aug;26(3):355-69).
• the present disclosure provides a method of treating osteoarthritis in a mammal in need thereof by administering a therapeutically effective amount of ⁇ Klotho protein or an active fragment thereof and a therapeutically effective amount of sTGF ⁇ -R2 protein or an active fragment thereof or a combination thereof to the mammal at a site within the mammal exhibiting osteoarthritis, wherein progression of the osteoarthritis is reduced compared to the untreated condition, or wherein cartilage at the site of the osteoarthritis is increased or regenerated or regrown compared to the untreated condition, or wherein inflammation is reduced compared to the untreated condition.
• An osteoarthritic site is one which exhibits symptoms of osteoarthritis.
• Osteoarthritis is the most common form of arthritis, affecting millions of people worldwide. It occurs when the protective cartilage that cushions the ends of your bones wears down over time.
• osteoarthritis can damage any joint, the disorder most commonly affects joints in your hands, knees, hips and spine. Symptoms of osteoarthritis include pain, stiffness, tenderness, loss of flexibility, grating sensation, bone spurs and swelling.
• Functional proteins as described herein can be the full length proteins or proteins which vary from the full length proteins but retain the activity in whole or in part of the full length protein.
• Figs. 1A-1F depict results of recapitulation of early-stage osteoarthritis phenotype in a rat model.
• Fig. ID depicts representative images from immunostaining detection of hypertrophic markers CollOa and
• FIGs. 2A-2C depict results of in vivo sTGF ⁇ R2 and ⁇ Klotho treatment inhibiting chondrocytes hypertrophy and promoting chondrocytes marker upregulation.
• FIG. 2B depicts a schematic of the time course for the osteoarthritis experiments.
• Female rats undergoing papain mediated osteoarthritis were either sacrificed after 4 weeks (OAC) or injected intra-articularly with AAVDJ-GFP (SHAM) or
• the thickness was determined by measuring the condyle cartilage at three different positions throughout the cartilage area. Quantification performed using
• Figs. 3 A-3C are directed to experiments where sTGF ⁇ R2 and ⁇ Klotho have been delivered by AAV-DJ serotype.
• Figs. 4A-4F depict images that show sTGF ⁇ R2 and ⁇ Klotho intra-articular injection promotes ECM repair and avoids apoptosis.
• KT treated knees show a complete cartilage structure with high safranin-0 staining.
• Fig. 4D depicts representative images from immunostaining detection of hypertrophic markers CollOa and
• Fig. 4E depicts representative images from immunostaining detection
• Fig. 4F depicts results of joint osteoarthritis grade in rats based on the OARSI scoring system
• Figs. 5A-5E depict results of sTGF ⁇ R2 and ⁇ Klotho inhibiting osteoarthritis-related immune response in vivo.
• Fig. 5A depicts a barplot of the statistical enrichment scores from common DE genes between [KT vs SHAM] and [HC vs OAC] (genes that shared common change in KT and HC, but behave differently in the other groups) according to Gene Ontology enrichment analysis. Only Biological Process terms with FDR (false discovery rate) ⁇ 0.01 were shown in the plot.
• Fig. 5B depicts a barplot of the statistical enrichment scores from KT vs SHAM DE genes
• Fig. 5C depicts a heatmap of gene expression for downregulated DE genes that shared common change in KT and HC, but behave differently in the other two groups. Colors indicated the gene-wise relative expression values across conditions
• FIG. 5D depicts gene expression plots of selected genes (from
• FIG. 5E depicts gene expression plots of Nos2 (from Fig. 7). Gene expression was normalized into FPKM values (Fragments Per
• HC blue
• OAC red
• SHAM green
• KT purple
• Figs. 6A-6D depict results of in vitro recovery of chondrocyte markers by sTGF ⁇ R2 and ⁇ Klotho.
• Fig. 6B depicts representative immunostaining images of Sox9 and Col2a from chondrocytes used in co-culture experiments
• Fig. 6D depicts that immunostaining quantification of
• Sox9, Col2a and EdU showed chondrocyte homeostasis improvement in those treated with sTGF ⁇ R2 and ⁇ Klotho recombinant proteins. Quantification performed by using Fiji software
• Fig. 7 depicts a heatmap that shows that sTGF ⁇ R2 and ⁇ Klotho prevent the activation of immune response mechanisms related to osteoarthritis.
• Heatmap of gene expression for DE genes differentially expressed between SHAM and KT (but were not differentially expressed between
• Row dendrogram showed the hierarchical clustering result based on the similarity of gene expression profile.
• Fig. 8 depicts data related to Grade (depth) of osteoarthritis in a mouse knee treated with the gene therapy by systemic injection as described herein.
• Fig. 9 depicts data related to Stage (extent) of osteoarthritis in a mouse knee treated with the gene therapy by systemic injection as described herein.
• Group mean +/- SEM.
• the 2-month post papain control (Group 2) exhibited the highest mean OA stage; a similar mean stage was observed in the STGFbR2 + FGF21(Group 4).
• a lower mean OA stage was observed in the 1- month post papain control (Group 1) compared to the 2 month control (Group 2).
• the lowest mean OA stage was observed in the STGFbR2 + ⁇ Klotho (Group 3), exhibiting a reduction in lesion severity when compared to the two control groups (Groups 1 and 2).
• Fig. 10 depicts data related to Score (grade x stage) of osteoarthritis in a mouse knee treated with the gene therapy by systemic injection as described herein.
• Group mean +/- SEM.
• the 2- month post papain group (Group 2) and the STGFbR2 + FGF21 (Group 4) exhibited the highest scores; no difference was observed between these groups.
• the 1-month post papain group (Group 1) exhibited the highest scores; no difference was observed between these groups.
• Fig. 11 depicts data related to meniscal fibrillation of osteoarthritis in a mouse knee treated with the gene therapy by systemic injection as described herein.
• FGF21 (Group 4); Group 4 exhibited the highest severity of meniscal fibrillation scores. Meniscal fibrillation was absent in the STGFbR2 + ⁇ Klotho(Group 3).
• Fig. 12A depicts data showing gene expression of various genes in response to treatment with a combination of sTGF ⁇ R2 and ⁇ Klotho versus sTGF ⁇ R2 individually and ⁇ Klotho individually. The combination synergistically achieved greater treatment effect compared to individual treatment.
• Fig. 12B depicts gel data demonstrating that chondrocytes treated with both factors showed higher protein expression of ACAN that each factor separately.
• the present disclosure provides methods and compositions for treating or preventing osteoarthritis, such as occurs with articular cartilage, using a therapeutically effective amount of a combination of ⁇ Klotho protein or an active fragment thereof and Soluble Tumor Growth Factor ⁇ receptor 2 (sTGF ⁇ R2) protein or an active fragment thereof which is administered to a mammal in need thereof as soluble proteins or a vector which is used to express the soluble proteins at a site within the mammal exhibiting osteoarthritis.
• sTGF ⁇ R2 Soluble Tumor Growth Factor ⁇ receptor 2
• Tumor Growth Factor ⁇ receptor 2 (sTGF ⁇ R2) is administered with ⁇ Klotho protein to treat or prevent osteoarthritis in joints such as the knee where articular cartilage is present.
• Soluble Tumor Growth Factor ⁇ receptor 2 ( sTGF ⁇ R2) and ⁇ Klotho are administered separately or simultaneously such that the Soluble Tumor Growth Factor ⁇ receptor 2 (sTGF ⁇ 2) and ⁇ Klotho are both present at the site of administration.
• the ⁇ Klotho protein or an active fragment thereof and the sTGI ⁇ -R2 protein or an active fragment thereof are encoded by nucleic acids that are included in one or more vectors or combined into a single viral vector, such as an AAV, which are administered to treat or prevent osteoarthritis and/or diseases or conditions associated with osteoarthritis.
• 8TGF ⁇ R2 acts to inhibit TGF ⁇ thereby inhibiting osteophyte formation despite increasing proteoglycans degradation (see Scharstuhl A, Glansbeek HL, van
• TGF ⁇ pathway regulates cartilage homeostasis in such a way that its balance and downstream effectors are essential for cartilage maintenance.
• TGF ⁇ has been considered as essential for the cartilage formation due to its role in chondrocyte proliferation and maturation, avoiding chondrocyte hypertrophy (Yang X, Chen L,
• soluble TGF ⁇ R2 (sTGF ⁇ R2), which lacks the membrane binding domain and has high affinity for TGF- ⁇ and ⁇ 3 (De Crescenzo G, Pham PL, Durocher
• TGF- ⁇ Transforming Growth Factor-beta
• ⁇ Klotho inhibits or prevents extracellular matrix (ECM) degradation (see Chuchana P, Mausset-Bonnefont A-L, Mathieu M, Espinoza F, Teigell M, Toupet
• Klotho codifies for a type I membrane-bound protein whose extracellular domain is released to the circulation by a 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, ⁇ Klotho regulates surface glycoproteins such as ion channels, the insulin-like growth factor 1 (IGF-l)/insulin and
• a method of treating osteoarthritis in a mammal in need thereof including administering a therapeutically effective amount of a combination of ⁇ Klotho protein or an active fragment thereof and sTGF ⁇ -R2 protein or an active fragment thereof to the mammal at a site within the mammal exhibiting osteoarthritis, wherein progression of the osteoarthritis is reduced compared to the untreated condition, or wherein cartilage at the site of the osteoarthritis is increased or regenerated or regrown compared to the untreated condition, or wherein inflammation is reduced compared to the untreated condition.
• the mammal is a dog or a human.
• the ⁇ Klotho protein or an active fragment thereof is administered as a soluble protein and the sTGF ⁇ -R2 protein or an active fragment thereof is administered as a soluble protein.
• the ⁇ Klotho protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection and the sTGF ⁇ -R2 protein or an active fragment thereof is administered as a soluble protein by intra-articular cartilage injection.
• a vector including a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the ⁇ TGF ⁇ -
• a vector including a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered by intra-articular cartilage injection and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the sTGF ⁇ -R2 protein or an active fragment thereof.
• a first vector including a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second vector including a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the sTGF ⁇ -R2 protein or an active fragment thereof.
• a first vector including a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second vector including a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered by intra-articular cartilage injection and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the sTGF ⁇ -R2 protein or an active fragment thereof.
• the vector is a recombinant virus.
• the vector is a parvovirus.
• the vector is an AAV vector.
• the AAV vector is AAV-DJ.
• the vector is an AAV vector serotyped for AAV1, AAV2, AAV3, AAV4, AAV5,
• the vector infects mesenchymal cells at the site of the osteoarthritis.
• the first vector and the second vector are a recombinant virus.
• the first vector and the second vector are a parvovirus.
• the first vector and the second vector are an AAV vector.
• the first vector and the second vector are an AAV vector.
• the first vector and the second vector are an AAV vector serotyped for AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
• the first vector and the second vector infect mesenchymal cells at the site of the osteoarthritis.
• the ⁇ Klotho protein or active fragment thereof and the sTGF ⁇ -R2 protein or active fragment thereof are human proteins.
• R2 protein or active fragment thereof are canine proteins.
• the ⁇ Klotho protein and the sTGF ⁇ -R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine and porcine proteins.
• the ⁇ Klotho protein has at least 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 99.5% or 100% sequence identity to sequence to the amino acid sequence of an ⁇ Klotho protein corresponding to SEQ ID NO: 1 as follows:
• the nucleic acid sequence encoding an ⁇ Klotho protein has at least 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 99.5% sequence or 100% sequence identity to the nucleic acid sequence encoding ⁇ Klotho protein corresponding to SEQ ID NO: 2 as follows:
• sTGF ⁇ -R2 protein has at least 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 99.5% sequence identity or 100% sequence identity to the amino acid sequence of sTGF ⁇ R2 receptor protein corresponding to SEQ ID NO: 3 as follows where the sTGFbR2 AA sequence is bolded sequence in the IGG PC domain and
• MGRGLLRGLWPLHIVLWTRIAST is the secretion signal:
• the nucleic acid sequence encoding an sTGF ⁇ -R2 protein has at least 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 99.5% sequence or 100% sequence identity to the nucleic acid sequence encoding sTGPp-R2 protein corresponding to SEQ ID NO: 4 as follows:
• the sTGF ⁇ -R2 protein and/or the ⁇ Klotho protein is an Fc fusion protein including an Ig Fc domain.
• the Ig Fc domain is selected from the group consisting of a human, a canine, a feline, a bovine, an ovine, a caprine, an equine, a murine, and a porcine Fc or a subtype thereof, including IgGl, IgG2a, IgG2b,
• the Ig Fc domain is a human Ig Fc domain that has at least 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 99.5% sequence identity or
• the Ig Fc domain is a mouse Ig Fc domain that has at least 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 99.5% sequence identity or
• the Ig Fc domain is a dog Ig Fc domain that has at least 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 99% sequence identity or 99.5% sequence identity or
• amino acid sequences having the described percent homology to the ⁇ Klotho protein amino acid sequence or the sTGF ⁇ -R2 protein amino acid sequence can be determined by obtaining the crystal structure for the ⁇ Klotho protein or the sTGF ⁇ -R2 protein and determining the active site or sites responsible for binding or activity and determining percent homology structures which maintain useful binding or activity. Portions of proteins identified as inactive are suitable for amino acid substitution or modification or mutation to produce proteins having the claimed percent homology. Active portions may also be modified or substituted or mutated to the extent that useful binding or activity results. Ig Fc sequences having the desired percent homology can be determined in a similar manner.
• GHECOM open source
• LIGSTTEcsc SURFNET
• SiteHound ICM-PocketFinder
• SiteEngine Tel- Aviv University
• SVILP_Ligand Imperial College London
• Useful databases include sc-PDB (University of France), CASTp, Pocketome (Encyclopedia of conformational ensembles of all draggable binding sites that can be identified experimentally from co-crystal structures in the Protein Data Bank), PDBe motifs and Sites, LigASite, PROtein
• Useful Web services include 3DLigandSite (Imperial College of London), metaPocket, PockDrug (University
• aspects of the present disclosure provide a vector including a first nucleic acid sequence encoding an ⁇ Klotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble Transforming Growth Factor Beta Receptor ⁇ (sTGF ⁇ -R2) protein or an active fragment thereof.
• a first promoter is operably linked to the first nucleic acid sequence for expression of the ⁇ Klotho protein or an active fragment thereof in a mammalian cell
• a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGF ⁇ -R2 protein or an active fragment thereof in a mammalian cell.
• the first promoter and the second promoter are cell or tissue specific.
• the first promoter and the second promoter are constitutive or inducible.
• the present disclosure provides a pharmaceutical formulation including a vector including a first nucleic acid sequence encoding an ⁇ Klotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble Transforming Growth Factor Beta Receptor ⁇
• sTGF ⁇ -R2 (sTGF ⁇ -R2) protein or an active fragment thereof in a pharmaceutically acceptable excipient.
• the transcribed polynucleotide can have a sequence encoding a polypeptide, such as a functional protein, which can be translated into the encoded polypeptide when placed under the control of an appropriate regulatory region.
• a gene may comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding sequence and a 3' nontranslated sequence, such as a polyadenylation site.
• a chimeric or recombinant gene is a gene not normally found in nature, such as a gene in which, for example, the promoter is not associated in nature with part or all of the transcribed
• “Expression of a gene” refers to the process wherein a gene is transcribed into an
• RNA and/or translated into a functional protein are examples of proteins.
• Gene delivery or “gene transfer” refers to methods for introduction of recombinant or foreign DNA into host cells.
• the transferred DNA can remain non-integrated or preferably integrates into the genome of the host cell.
• Gene delivery can take place for example by transduction, using viral vectors, or by transformation of cells, using known methods, such as electroporation, cell bombardment.
• Transgene refers to a gene that has been introduced into a host cell.
• the transgene may comprise sequences that are native to the cell, sequences that do not occur naturally in the cell, or combinations thereof.
• a transgene may contain sequences coding for one or more proteins that may be operably linked to appropriate regulatory sequences for expression of the coding sequences in the cell.
• Transduction refers to the delivery of a nucleic acid molecule into a recipient host cell, such as by a gene delivery vector, such as rAAV.
• a gene delivery vector such as rAAV.
• transduction of a target cell by a rAAV virion leads to transfer of the rAAV vector contained in that virion into the transduced cell.
• Home cell or “target cell” refers to the cell into which the nucleic acid delivery takes place.
• “Functional protein” includes variants, mutations, homologues, and functional fragments of the full length proteins.
• One of skill will readily be able to construct proteins homologous to the full length proteins which retain the activity, in whole or in part, of the full length protein, based on the present disclosure.
• Vector refers generally to nucleic acid constructs suitable for cloning and expression of nucleotide sequences.
• a vector is a viral vector.
• the term vector may also sometimes refer to transport vehicles comprising the vector, such as viruses or virions, which are able to transfer the vector into and between host cells.
• 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,
• rAAV vectors can have one or preferably all wild type AAV genes deleted, but still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue and packaging of AAV virions.
• the ITR sequences may be wild type sequences or substantially identical sequences (as defined below) or may be altered by for example in insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional.
• “Therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as results directed at osteoarthritis and related diseases or conditions.
• a therapeutically effective amount of a parvoviral virion or pharmaceutical composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the parvoviral virion or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
• a therapeutically effective amount is also typically one in which any toxic or detrimental effects of the parvoviral virion or pharmaceutical composition are outweighed by the therapeutically beneficial effects.
• prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting osteoarthritis.
• a prophylactic dose may be used in subjects prior to or at an earlier stage of disease, and a prophylactically effective amount may be more or less than a therapeutically effective amount in some cases.
• Nucleic acid includes any molecule composed of or comprising monomeric nucleotides.
• the term “nucleotide sequence” may be used interchangeably with “nucleic acid” herein.
• a nucleic acid may be an oligonucleotide or a polynucleotide.
• a nucleic acid may be a
• a nucleic acid may be a gene.
• a nucleic acid may be chemically modified or artificial.
• Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA).
• Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule. Also, phosphorothioate nucleotides may be used.
• Nucleic acid construct is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology.
• a nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids, which are combined and juxtaposed in a manner, which would not otherwise exist in nature.
• a nucleic acid construct usually is a “vector”, i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.
• One type of nucleic acid construct is an “expression cassette” or “expression vector”.
• nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences.
• Expression cassettes or expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3’ transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements.
• a nucleic acid construct can also be a vector in which it directs expression or repression of a protein by operating as
• RNA instead of DNA.
• the nucleic acid construct can be mRNA or similar in which the cell or more specifically the ribosome would recognize and create many copies of the protein.
• the RNA can be in the form that acts through preventing the ribosome from creating protein. This can be done through mechanisms of RNAi or shRNA or miRNA or Pri-miRNA.
• repressing a known repressor of a target sequence can result in an increase in the target sequence through repression through the delivery of either mRNA (or similar) or shRNA (or similar) to regulate the target sequence. This can also be done through the vector that provides DNA that is expressed, such as when using AAV.
• operably linked refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
• a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
• a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
• Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.
• “Expression control sequence” refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked.
• An expression control sequence is “operably linked” to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence.
• an expression control sequence can include promoters, enhancers, internal ribosome entry sites
• first and second nucleic acid sequences encoding sTGF ⁇ -R2 protein and ⁇ Klotho are separated by a polycistronic clement.
• a polycistronic element is generally understood to describe a type of messenger RNA that can encode more than one polypeptide separately within the same RNA molecule.
• expression control sequence is intended to include, at a minimum, a sequence whose presence is designed to influence expression, and can also include additional advantageous components.
• leader sequences and fusion partner sequences are expression control sequences.
• the term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3’-end of a mRNA, which may be referred to as polyA sequences. It also can be designed to enhance mRNA stability.
• pA polyadenylation sequences
• Expression control sequences which affect the transcription and translation stability, e.g., promoters, as well as sequences which effect the translation, e.g., Kozak sequences, suitable for use in insect cells are well known to those skilled in the art.
• Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved.
• CAT catalase
• a better known version can replace the natural sequence for enhanced effect, such as taking the human or mouse secretion signal for TGFbR2 and fusing it to the dog version of the protein.
• Promoter or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent
• RNA polymerase RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g. attenuators or enhancers, but also silencers.
• a “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
• An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.
• a “tissue specific” promoter is only active in specific types of tissues or cells.
• the disclosure provides for the operable linking of nucleic acid constructs to a mammalian cell-compatible expression control sequence, e.g., a promoter.
• a mammalian cell-compatible expression control sequence e.g., a promoter.
• Many such promoters are known in the art (see Sambrook and Russell, 2001, supra).
• Constitutive promoters that are broadly expressed in many cell types, such as the CMV and hEfla promoter are disclosed. Variations of the full-length hEfla are also disclosed which are shorter but still provide effective constitutive expression.
• promoters that are inducible, tissue- specific, cell-type-specific, or cell cycle-specific.
• the nucleotide sequence encoding the porphobilinogen deaminase is operably linked to a liver-specific promoter.
• liver-specific promoters are particularly preferred for use in conjunction the non- erythroid deaminase.
• an expression control sequence for liver-specific expression are e.g. selected from the group consisting of an al-anti-trypsin
• AAT a thyroid hormone-binding globulin promoter
• an albumin promoter a thyroxin-binding globulin (TBG) promoter
• an Hepatic Control Region (HCR)-ApoCII hybrid promoter an HCR-hAAT hybrid promoter
• an AAT promoter combined with the mouse albumin gene enhancer (Ealb) element and an apolipoprotein E promoter.
• E2F promoter for tumour-selective, and, in particular, neurological cell tumour- selective expression (Parr et al., (1997) Nat. Med. 3:1145-9) or the IL-2 promoter for use in mononuclear blood cells (Hagenbaugh et al., (1997) J Exp Med; 185: 2101-10).
• 3 * UTR or “3 * non-translated sequence” (also often referred to as 3’ untranslated region, or 3’ end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof).
• the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).
• “Naturally occurring sequence” or “native sequence” as used herein refers to a polynucleotide or amino acid isolated from a naturally occurring source. Included within
• mutant sequence are recombinant forms of a native polypeptide or polynucleotide which have a sequence identical to the native form.
• “Mutant” or “variant” as used herein refers to an amino acid or polynucleotide sequence which has been altered by substitution, insertion, and/or deletion. In some embodiments, a mutant or variant sequence can have increased, decreased, or substantially similar activities or properties in comparison to the parental sequence.
• Percentage of sequence identity and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
• the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of
• HSPs high scoring sequence pairs
• Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• W wordlength
• E expectation
• BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the
• the degree of percent amino acid sequence identity can also be obtained by ClustalW analysis (version W 1.8) by counting the number of identical matches in the alignment and dividing such number of identical matches by the length of the reference sequence, and using the following default ClustalW parameters to achieve slow/accurate pairwise optimal alignments - Gap Open Penalty: 10; Gap Extension Penalty: 0.10; Protein weight matrix:
• Subject 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 may be a domesticated animal, such as a dog, a cat, a mouse, a cow, a sheep, a goat, a horse, or a pig.
• the mammal may be a human subject.
• the human is an adult patient. In some embodiments, the human is a pediatric patient.
• Foreign nucleic acids alternatively referred to as heterologous nucleic acids (i.e., those which are not part of a cell’s natural nucleic acid composition) may be introduced into a 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, conjugation and the like.
• methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like.
• Foreign nucleic acids may be delivered to a subject by administering to the subject, such as systemically administering to the subject, such as 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, a nucleic acid or vector including a nucleic acid as described herein.
• vector includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• Vectors used to deliver the nucleic acids to cells as described herein include vectors known to those of skill in the art and used for such purposes.
• Certain exemplary vectors include, among others, plasmids, lentiviruses, and adeno-associated viruses as is 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 comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
• plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
• viral vector is another type of vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus, e.g.
• Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
• Certain vectors are capable of autonomous replication in a 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
• certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as
• Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
• “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory elements) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
• Methods of non-viral delivery of nucleic acids or native DNA binding protein, native guide RNA or other native species include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in, e.g., U.S.
• Lipofection reagents are also available from commercial sources (e.g., TransfectamTM and LipofectinTM).
• Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
• the term native includes the protein, enzyme or guide RNA species itself and not the nucleic acid encoding the species.
• the gene therapy vectors for use in the methods herein are parvoviral vectors, such as animal parvoviruses, in particular dependoviruses such as infectious human or simian adeno-associated virus (AAV), and the components thereof (e.g., an animal parvovirus genome) for use as vectors for introduction and/or expression of the nucleotide sequences encoding a porphobilinogen deaminase in mammalian cells.
• parvoviral vectors such as animal parvoviruses, in particular dependoviruses such as infectious human or simian adeno-associated virus (AAV), and the components thereof (e.g., an animal parvovirus genome) for use as vectors for introduction and/or expression of the nucleotide sequences encoding a porphobilinogen deaminase in mammalian cells.
• Parvoviridae family are small DNA animal viruses.
• the family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects.
• Members of the subfamily Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirus.
• members of the Dependovirus are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture.
• the genus Dependovirus includes AAV, which normally infects humans (e.g., serotypes 1, 2, 3 A,
• AAV is a linear, single stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length.
• Inverted terminal repeats flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins.
• the VP proteins are highly repetitive DNA sequences that flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins.
• the terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex.
• Rep genes i.e., Rep78 and Rep52
• both Rep proteins have a function in the replication of the viral genome.
• a splicing event in the Rep ORF results in the expression of actually four
• Rep proteins i.e., Rep78, Rep68, Rep52 and Rep40.
• Rep78, Rep68, Rep52 and Rep40 unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for
• a “recombinant parvoviral” or “AAV vector” or “rAAV vector” herein refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest or
• transgenes that are flanked by at least one parvoviral or AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that is expressing AAV rep and cap gene products
• an rAAV vector When an rAAV vector is incorporated into a larger nucleic acid construct (e.g. in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), then the rAAV vector is typically referred to as a “pro-vector” which can be “rescued” by replication and encapsulation in the presence of AAV packaging functions and necessary helper functions.
• the invention relates to a nucleic acid construct comprising a nucleotide sequence encoding a porphobilinogen deaminase as herein defined above, wherein the nucleic acid construct is a recombinant parvoviral or AAV vector and thus comprises at least one parvoviral or AAV ITR.
• the nucleotide sequence encoding the porphobilinogen deaminase is flanked by parvoviral or AAV ITRs on either side.
• AAV is able to infect a number of mammalian cells. See, e.g., Tratschin et al., (1985)
• AAV differs among serotypes. See, e.g., Davidson et al. (2000) Proc. Natl. Acad. Sci. USA,
• AAVs are highly prevalent within the human population. See Gao, G., et al., (2004) J
• AAV9 has been demonstrated to cross the blood-brain barrier. See Foust, K.D., et al.,
• the AAV VP proteins are known to determine the cellular tropicity of the AAV virion.
• the VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes.
• the ability of Rep and ITR sequences to crosscomplement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid proteins of one serotype (e.g., AAVS) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2).
• pseudotyped rAAV particles are a part of the present invention.
• a pseudotyped rAAV particle may be referred to as being of the type “x/y”, where “x” indicates the source of ITRs and “y” indicates the serotype of capsid, for example a 2/5 rAAV particle has ITRs from AAV2 and a capsid from AAV5.
• Modified “AAV” sequences also can be used in the context of the present disclosure, e.g. for the production of rAAV vectors in insect cells. Such modified sequences e.g.
• nucleotide and/or amino acid sequence identity include 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 and/or amino acid sequence identity (e.g., a sequence having from about 75% to about 99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10,
• AAV11, AAV12, AAV2.5, AAvDJ, AAVihlO.XX ITR, Rep, or VP can be used in place of wild-type AAV ITR, Rep, or VP sequences.
• Preferred adenoviral vectors are modified to reduce the host response. See, e.g., Russell (2000) J. Gen. Virol. 81:2573-2604; US patent publication no. 20080008690; and Zaldumbide et al. (2008) Gene Therapy 15(4):239-46; all publications incorporated herein by reference.
• regulatory element is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences).
• promoters e.g. promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences).
• ITR internal ribosomal entry sites
• Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
• tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes).
• Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
• a vector may comprise one or more pol ⁇ promoter (e.g., 1, 2, 3, 4, 5, or more pol ⁇ promoters), one or more pol ⁇ promoters (e.g., 1, 2, 3, 4, 5, or more pol ⁇ promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.
• pol ⁇ promoters include, but are not limited to, U6 and HI promoters.
• pol ⁇ promoters include, but are not limited to, the retroviral Rous sarcoma virus
• RSV LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer; see, e.g., Boshart et al, (1985) Cell 41:521-530) the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter and Pol ⁇ promoters described herein.
• regulatory element are also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Takebe, Y. (1988)
• a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein
• CRISPR clustered regularly interspersed short palindromic repeats
• a terminator sequence includes a section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription. This sequence mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger processes which release the mRNA from the transcriptional complex. These processes include the direct interaction of the mRNA secondary structure with the complex and/or the indirect activities of recruited termination factors. Release of the transcriptional complex frees RNA polymerase and related transcriptional machinery to begin transcription of new mRNAs. Terminator sequences include those known in the art and identified and described herein.
• the one or more gene delivery vectors can be in the form of a medicament or a pharmaceutical composition and may be used in the manufacture of a medicament or a pharmaceutical composition.
• the pharmaceutical composition may include a pharmaceutically acceptable carrier.
• the carrier is suitable for parenteral administration.
• the carrier is suitable for intravenous, intra-articular, intraperitoneal or intramuscular administration.
• Pharmaceutically acceptable carrier or excipients are described in, for example, Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro
• Exemplary pharmaceutical forms can be in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluids.
• a solid carrier may be used such as, for example, microcarrier beads.
• compositions are typically sterile and stable under the conditions of manufacture and storage.
• Pharmaceutical compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to delivery of the gene therapy vectors.
• the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
• the vectors of the present disclosure may be administered in a time or controlled release formulation, for example in a composition which includes a slow release polymer or other carriers that will protect the compound against rapid release, including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers may for example be used, such as ethylene vinyl acetate, polyanhydridcs, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG).
• the gene therapy vectors can be administered pixieerally, such as by intravenous, intra-articular, intraperitoneal, subcutaneous, intramuscular administration, limb perfusion or combinations thereof.
• the administration can be systemic, such that the gene delivery vectors are delivered through the body of the subject.
• the gene delivery vectors can be administered directly into the targeted tissue.
• the gene delivery vectors can be administered locally, such as by a catheter.
• the route of administration can be determined by the person of skill in the art, taking into consideration, for example, the nature of target tissue, gene delivery vectors, intended therapeutic effect, and maximum load that can be administered and absorbed by the targeted tissue(s).
• an effective amount, particularly a therapeutically effective amount, of the gene delivery vectors are administered to a subject in need thereof.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment or amelioration of osteoarthritis.
• An effective or therapeutically effective amount of vector may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the viral vector to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
• a range for therapeutically or prophylactically effective amounts of a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition may be from lxlO 11 and lxlO 14 genome copy (gc) /kg or lX10 12 and lxlO 13 genome copy (gc) /kg.
• dosage values may vary with the severity of the condition to be alleviated. The dosage may also vary based on the efficacy of the virion employed. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.
• the tissue target may be specific, for example the articular cartilage associated with osteoarthritis.
• the effective dose range for small animals may be between lX10 12 and lxlO 13 genome copy (gc) /kg, and for larger animals (cats or dogs) and for human subjects, between lxlO 11 and lX10 12 gc/kg, or between lxlO 11 and lxlO 14 genome copy (gc) /kg.
• the gene delivery vectors can be administered as a bolus or by continuous infusion over time. In some embodiments, several divided doses can be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the gene delivery vectors can be administered daily, weekly, biweekly or monthly. The duration of treatment can be for at least one week, one month, 2 months, 3 months, 6 months, or 8 month or more. In some embodiments, the duration of treatment can be for up to 1 year or more, 2 years or more, 3 years or more or indefinitely.
• a therapeutically effective amount of ⁇ Klotho protein or an active fragment thereof and sTGF ⁇ -R2 protein or an active fragment thereof is administered to the subject to treat osteoarthritis, such as exhibited by articular cartilage.
• Methods described herein treats or prevents disorders associated with osteoarthritis or symptoms of osteoarthritis or otherwise improves recovering of articular cartilage structure and function.
• the amelioration of osteoarthritis provided by the administration of ⁇ Klotho protein or an active fragment thereof and sTGF ⁇ -R2 protein or an active fragment thereof directly or through a gene therapy method as described herein is characterized by a reduction of symptoms in a subject compared to an untreated subject.
• a gene therapy method or the use of a nucleic acid vector as described above is provided for use in the treatment or prevention in a subject of osteoarthritis.
• the administration of ⁇ Klotho and sTGF ⁇ R2 cooperatively inhibits or prevents osteoarthritis progression such as by downregulating the immune response and promoting joint tissue homeostasis and repair.
• the present disclosure provides for an animal model of osteoarthritis for use in experiments described herein.
• Osteoarthritis was mimicked by the intra-articular injection of papain, a chemically induced model that promotes proteoglycans degradation disrupting cartilage microarchitecture and affecting the integrity of the knee joints (see Pritzker KP. Animal models for osteoarthritis: processes, problems and prospects. Ann Rheum Dis. 1994 Jun;53(6):406-20).
• This enzyme does not have a direct impact on collagen and chondrocytes; thereby, it does not impair the regeneration mechanisms of the cartilage that may be promoted by tested treatments.
• the model recapitulated several osteoarthritis phenotypes associated with the pathology in animals and humans. For instance, the loss of ECM homeostasis caused by proteoglycan-degrading enzymes such as the MMP13 is one of the main pathological features described in OA patients (see Troeberg
• Rat knee joint was analyzed four weeks after the papain injection.
• the safranin-0 staining showed clear signs of early-stages of osteoarthritis according to the normalized Osteoarthritis
• the rats (here on, Osteoarthritis Control group, OAC) showed a grade 2 osteoarthritis (see
• Fig. 1A as defined by the parameters analyzed.
• the safranin-0 staining showed diminished cartilage thickness with discontinued fibrillar surface and cellular clusters within the cartilage (see
• Fig. IB The osteoarthritis grade in these samples was further supported by tunel and immunostaining analysis that demonstrated not only the presence of cellular death (see Fig. 1C), but also of hypertrophic chondrocytes within the joint according to higher levels of CollOa and
• Runx2 (see Fig. ID) together with the downregulation of the chondrocyte marker Sox9 (see Fig.
• MMP13 within the matrix indicates cartilage damage and loss of joint function (see Xie Y, Mustafa
• Nuclear matrix metalloproteinases functions resemble the evolution from the intracellular to the extracellular compartment.
• each factor or active fragment thereof may be encoded by a separate nucleic acid and be provided in a separate vector or each factor may be encoded by a single nucleic acid and be provided in a single vector.
• the single nucleic acid may be expressed to produce separate soluble factors or may be expressed as a fusion protein of the soluble factors.
• aspects of the present disclosure contemplate directly injecting or otherwise delivering or administering the soluble factors ⁇ Klotho (or an active fragment thereof) and sTGF ⁇ R2 (or an active fragment thereof) to a patient in need of treatment.
• the ⁇ Klotho (or an active fragment thereof) and sTGF ⁇ R2 (or an active fragment thereof) can be administered separately in formulations, such as one after the other in series or can be administered together, such as co-administered in the same formulation.
• the nucleic acid encoding ⁇ Klotho (or an active fragment thereof) and the nucleic acid encoding sTGF ⁇ R2 (or an active fragment thereof) can be administered separately in separately formulations, such as one after the other in series or can be administered together, such as co-administered in the same formulation.
• the dosage of ⁇ Klotho (or an active fragment thereof) and sTGF ⁇ R2 (or an active fragment thereof) is in the range of 1X10 12 to 100X10 12 GC (AAV-DJ).
• An exemplary dose of 2.5xl0 12 GC (AAV-DJ) can be injected at the desired site such as the knee in
• both ⁇ Klotho and sTGF ⁇ R2 will be released by adjacent mesenchymal cells localized in the joint, exerting their effect in the whole joint.
• rats treated with papain were allowed to develop early-stage osteoarthritis during four weeks and then were treated by intra-articular injection with either AAV-DJ-GFP (SHAM group) or AAV-DJ- ⁇ Klotho and -sTGF ⁇ R2 (here on, KT group) (schematic representation can be found in Fig. 2B).
• AAV-DJ-GFP SHAM group
• AAV-DJ- ⁇ Klotho and -sTGF ⁇ R2 here on, KT group
• the rats injected with AAV-DJ-GFP showed an even greater deterioration of their cartilage six weeks later.
• the safranin-O, Col2a and ACAN staining showed not only a clear erosion and loss of the cartilage structure but also a calcification of the matrix, as demonstrated by the drastic down regulation of the ECM components in the remaining fragments (see Figs. 4 A and 4B, and
• the rats treated with AAV-DJ- ⁇ Klotho and -sTGF ⁇ R2 showed a greatly improved phenotype after 6 weeks.
• Separate vectors were used to make separate AAV-DJ viruses.
• a total of 2.5xl0 12 GC were injected in the joint, 50% sTGF ⁇ R2 and 50% ⁇ Klotho.
• the intra- articular injection of AAV expressing ⁇ Klotho and sTGF ⁇ R2 not only avoided the release of MMP13 to the ECM, but also promoted the maintenance of the cartilage thickness.
• Fig. 2C Fig. 2C and structure.
• First the superficial zone, where the cells are arranged in horizontal clusters parallel to the articular surface, organized in strings, pairs and single cells.
• Col2a and ACAN positive staining supports chondrocytes functional recovery with regeneration of the ECM components within the joint (see Fig. IB and Fig. 2A).
• ECM degradation supports the increase of Col2a, ACAN and Safranin-0 staining after KT treatment. This was further assessed in tissue section analysis. The data not only shows a complete absence of apoptotic cells within the joint (see Fig. 4C), but also the restoration of the hypertrophic markers distribution. Contrary to OAC and SHAM groups, in the KT treated joints, CollOa and
• Runx2 positive cells are mostly located in the lower levels of the cartilage layer corresponding to the regular hypertrophic layer of the cartilage, similar to HC group (see Fig.4D). Also, the absence of the proteolytic enzyme MMP13 was found within the ECM in HC and KT treated knees (see
• the OARSI classification indicates that the rats treated with ⁇ Klotho and sTGF ⁇ R2 recovered from a grade 2 to grade 1 osteoarthritis, while those treated with AAV-DJ-GFP progressed further to grade 4 osteoarthritis (see Fig. 4F).
• TGF- ⁇ /Smad signaling also contributes to osteoarthritis development and progression (see
• the use of both ⁇ Klotho and sTGF ⁇ R2 contributes to ECM recovery, for example, by balancing anabolic and catabolic pathways.
• the TGF ⁇ pathway is considered a reparative mediator by stimulating chondrocyte proliferation.
• the use of sTGF ⁇ R2 to sequester TGF ⁇ reduces the catabolic pathways while enhancing its anabolic effects.
• osteoarthritis 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 significant cartilage loss and joint space narrowing of osteoarthritic joints (Sokolove J, Lepus CM. Role of inflammation in the pathogenesis of osteoarthritis: latest findings and interpretations. Ther Adv
• cartilage tissues were isolated from all the groups for RNA-seq analysis.
• RNA-seq analysis revealed differentially expressed (DE) genes in the KT group when compared to OAC and SHAM group. Specifically, 489 genes were differentially expressed in KT vs SHAM, and 156 of them show a similar expression pattern between KT and HC. Gene Ontology (GO) analysis indicated that among these differentially expressed genes, those involved in inflammatory response and immune response exhibited the most dramatic effect upon KT treatment (see Figs.
• chondrocytes secrete proinflammatory cytokines under pathological conditions such as osteoarthritis (see Akkiraju H, Nohe A. Role of
• the data demonstrate that four weeks after the papain treatment the chondrocytes already show upregulation of pro-inflammatory cytokines and immune response related factors.
• ⁇ Klotho and sTGF ⁇ R2 treatment not only downregulated the expression of some of those already expressed genes, but also avoided the posterior upregulation of other immune response factors demonstrating the role of TGF ⁇ in inflammation during osteoarthritis.
• TGF ⁇ induces synovial lining cells to produce inflammatory factors which can further stimulate hyaline chondrocytes hypertrophy (see Scanzello CR, Goldring SR. The role of synovitis in osteoarthritis pathogenesis. Bone. 2012 Aug;51(2):249-57).
• TGF ⁇ signaling blockage significantly attenuated the synovial thickening implicated in the pathogenesis of osteoarthritis (see Scharstuhl A, Vitters
• soluble Klotho modulates the PBK/Akt and Wnt ⁇ -catenin pathways, which are involved in cellular inflammatory responses.
• Different studies demonstrated how recombinant Klotho treatment was able to reduce cytokine levels implicated in kidney and cardiac disease see Zhao Y, Banerjee S, Dey N, LeJeune WS, Sarkar PS, Brobey R, et al. Klotho
• the cooperative activity of both ⁇ Klotho and sTGF ⁇ R2 reduced the osteoarthritis-related inflammatory response.
• NO nitric oxide
• aspects of the present disclosure are directed to the use of ⁇ Klotho (or an active fragment thereof) and sTGF ⁇ R2 (or an active fragment thereof) as soluble factors directly or via gene therapy to avoid or lower the subsequent destructive processes induced by the proinflammatory response.
• the KT treatment prevented the upregulation of this enzyme which drastically increased in the SHAM animals any time after the AAV injection.
• AAV-mediated ⁇ Klotho and sTGF ⁇ R2 expression avoided cartilage degradation by diminishing ⁇ L- ⁇ -induced NO production throughout the reduction of Elm and
• ⁇ Klotho reduces oxidative stress and downregulates apoptosis upon KT treatment.
• the articular chondrocytic phenotype is characterized by the expression of cartilage-specific extracellular matrix components, predominantly Col2a, and the cartilage-specific transcription factor Sox9.
• Sox9 is required for the commitment of mesenchymal cells toward the chondrogenic 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 is highly dependent on the culture conditions.
• a method for the regrowth of cartilage by administering ⁇ Klotho or an active fragment thereof and sTGF ⁇ R2 or an active fragment thereof or administration of the genes in a vector for expression, such as AAV-DJ- ⁇ Klotho and AAV-DJ- sTGF ⁇ R2 treatment.
• the human articular chondrocytes were also treated in vitro using ⁇ Klotho and sTGF ⁇ R2 during 10 days.
• the results showed the same clear improvement demonstrated by the induction of Sox9 and Col2a protein expression, and the enhancement of cell proliferation (see
• a method for treating human hyaline cartilage by administering ⁇ Klotho or an active fragment thereof and sTGF ⁇ R2 or an active fragment thereof or administration of the genes in a vector for expression, such as AAV-DJ- ⁇ Klotho and AAV-DJ- sTGF ⁇ R2 treatment. Accordingly, ⁇ Klotho and sTGF ⁇ R2 are administered to maintain chondrocytic phenotype in humans.
• Human articular cartilage was obtained from healthy donors following informed consent for use in medical research, and rat articular cartilage was removed from the femoral and tibial condyles from a healthy rat under sterile conditions. Chondrocytes isolation and cultured was performed as described previously (see Gosset M, Berenbaum F, Thirion S, Jacques C. Primary culture and phenotyping of murine chondrocytes. Nat Protoc. 2008;3(8): 1253-60).
• Human fibroblast (IMR90) were grown in basic culture medium at 37 °C and 5% CO2 conditions. Rat mesenchymal cells were isolated from joint capsule connective tissues. Briefly, connective tissues were enzymatically digested (as described (Yu G, Wu X, Kilroy G, Halvorsen
• the AAV plasmids were constructed following standard cloning techniques. a-Klotho and
• STGF ⁇ R2 were PCR amplified using primers as follows. a-Klotho-fwd-5 ' - g g ggg g C ⁇ -Klotho-rev-cctgaacgtctcgct accttaTT AC 1TAT AAC 11 Cl Cl GGCC sTGF ⁇ R2-fwd-5 ' -cctgaacacctgcaacgggcctgcacc AT -3’ the uppercase is the overlap to the secretion signal of TGF ⁇ 2 receptor, the bold is the Aarl recognition site that creates a Notl overhang.
• the bold is the overlap to the mouse Igg2a FC region and the lower case matches the extracellular domain of TGF ⁇ R2 sequence used for overlap PCR.
• Igg2a-Rev 5’- the bold is the Aarl recognition site that creates a Nhel overhang.
• AAVs were prepared using 293AAV cells (Cell Biolabs, Inc.) as described with minor modifications (see Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno- associated viral vectors. Nat Pro toe. 2006;l(3):1412-28). Briefly, cells were transfected using calcium phosphate and the virus purified by CsCl gradient. Virus titer was determined via qPCR using primers; rTR-F:5’ T 3’ and ITR-R: 5’
• HS healthy control group
• OAC osteoarthritis group control
• KT sTGF ⁇ R2 group
• SHAM 4 week osteoarthritis SHAM group
• OAC, KT and SHAM rats were treated with papain/cysteine.
• OAC rats were sacrificed 4 weeks after the last papain/cysteine injection to determine the grade of osteoarthritis reached at that point.
• the other 2 groups were subjected to intra- articular
• AAV treatment AAV-DJ-GFP was injected to the SHAM group, and AAV-DJ- ⁇ Klotho+AAV- DJ- sTGF ⁇ R2 to the KT group.
• a total number of 2.5xl0 12 GC was injected per knee in 50 ⁇ 1 of
• RNA concentrations were determined using Qubit Fluorometer 2.0.
• the whole knee joint was prepared for histology as described in Kawamoto and Shimizu,
• 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).
• (OARSI) scoring System following the advanced methodology(23): A grade of 0 was received for normal and healthy cartilage; grade 1 was applied when the cartilage surface was intact but contained abrasion areas, hypertrophy and cellular clusters; grade 1.5 refers to a grade 1 that includes cell death; grade 2 cartilage presented discontinued fibrillar surface; grade 2.5 consist on a grade 2 that include the loss of matrix shown by less than 1/3 of Safranin-0 staining; grade 3 was considered when fissures appeared up to the mid zone and Safranin-0 stained less than 2/3 of the cartilage; grade 3.5 presented deeper fissures into the mid zone; grade 4 refers to matrix loss by delamination of the superficial zone; grade 4.5 showed excavation into the mid zone; grade 5 cartilage showed completely eroded unmineralized cartilage; grade 5.5 showed growth of hypertrophy cartilage after the erosion; and 6, the higher grade, indicates the more severe cartilage damage when deformation of the condyle appears.
• the thicknesses of the whole condylar cartilage were measured using the image analysis software Image J. The samples were evaluated by 2 blinded investigators and considering three different areas along the cartilage length.
• the apoptosis detection was carried out on rat knee sections using In situ cell death detection AP kit (Roche) according to the manufacturer’s protocol.
• Collagen ⁇ (NeoMaikers) at a 1:100 (v/v) dilution, Runx2 (Santa Cruz Biotechnology) at a 1:100 (v/v) dilution, MMP13 (Abeam) at a 1:100 (v/v) dilution, MMP3 (Abeam) at a 1:100 (v/v) dilution, Sox9 (Abeam) at a 1:100 (v/v) dilution, Collagen X (Abeam) at a 1:50 (v/v), Ki67
• Luc or AAV-DJ empty vector, negative control.
• the luciferase was detected 2 weeks after injection using an IVIS Kinetic 2200 (Caliper Life Sciences). 50 mg/kg D-Luciferin (Biosynth) was injected intra-articular and intraperitoneal. Imaging was captured 10 min after the D-luciferin injection.
• GFP positive cells were detected using a FACS Canto ⁇ (BD Biosciences) after filtering
• Quantification data is expressed as averages ⁇ s.e.
• Statistical significance (*P values) was determined by an unpaired, two-tailed Student’s /-test with Welch’s correction (there is no assumption of an equal s.d. in each group). All analyses were performed with Prism 7 software from GraphPad (San Diego, CA, USA). Statistical significance was determined to be P ⁇ 0.05,
• Osteoarthritic (OA) lesions were created by intra-articular injection of papain as described herein and primarily consisted of an intact cartilage surface with superficial fibrillation, chondrocyte death/loss and proliferation, edema, and/or loss of proteoglycan matrix in the superficial zone. Occasionally, deeper fibrillations, abrasions, and fissures were observed extending into the mid zone. Additionally, superficial fibrillations were observed along the surface of the menisci. Fibrillations were characterized by small cracks and discontinuities of the cartilage matrix in the superficial zone or the meniscal surface.
• Chondrocyte death/loss was characterized by the absence of chondrocytes or “ghost” chondrocytes present in the superficial and mid zones of cartilage. Chondrocyte proliferation was characterized by increased numbers of chondrocytes, often disorganized, within the superficial and mid zones of cartilage. Edema was characterized by increased clear fluid surrounding chondrocytes of the superficial and mid zones of the cartilage. Loss of proteoglycan matrix was characterized by decreased or absent cationic staining (red; Safranin
• loss of matrix could be present directly adjacent to viable chondrocytes or in areas of chondrocyte loss and was typically accompanied by other lesions as described above.
• Abrasions were characterized by focal loss of the superficial zone leaving a roughened surface.
• Fissures were characterized by vertical matrix separation extending into the mid zone cartilage.
• Viral vectors comprising nucleic acids encoding sTGFbR2 and FGF21 were systemically injected into rats to determine gene therapy treatment of osteoarthritis in rat knees.
• OA grade was determined based on the depth (superficial, mid, deep, or with bone involvement) of the most severe lesions observed in the sample.
• a slight reduction in grade was observed in the sTGFbR2 + ⁇ Klotho treated group (Group 3) when compared to the 2-month post papain control group (Group 2).
• OA stage was determined based on the total extent of the cartilage affected by the OA lesions. Amongst group trends were similar to those observed for OA grade, however the lowest mean score for OA stage was observed in the sTGFbR2 + ⁇ Klotho treated (Group 3).
• OA score was determined by multiplying the grade and stage for a total OA value.
• the 2- month post papain group (Group 2) and the sTGFbR2 + FGF21 treated group (Group 4) exhibited identical as well as the highest scores.
• the 1 -month post papain group (Group 1) had lower scores compared to the 2-month post papain group (Group 2).
• the sTGFbR2 + ⁇ Klotho treated group (Group 3) exhibited the lowest scores, indicating a reduction in lesion severity compared to the two control groups (Groups 1 and 2).
• KT was first analyzed by using an osteoarthritis in vitro model using high TGFbl concentration.
• the results analyzed by qPCR showed how the combination of both soluble factors synergistically favors the inhibition of hypertrophic markers and ECM proteolytic enzymes when compared to the single factor treatments (Fig. 12A, KT versus K versus T).
• chondrocytes treated with both factors showed higher protein expression of ACAN than ⁇ KLOTHO or sTGFbR2 (Fig. 12B).
• Fig. 12C describes the time line of injection.
• Embodiments of the present disclosure are directed to a method of treating osteoarthritis in a subject in need thereof, the method including the step of administering to the subject a first viral vector including a first nucleic acid sequence encoding an sTGF ⁇ -R2 protein or an active fragment thereof; and a second nucleic acid sequence encoding an ⁇ Klotho protein or an active fragment thereof, thereby treating osteoarthritis in the subject.
• the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element.
• the polycistronic element is an IRES or 2A sequence.
• Embodiments of the present disclosure are directed to a method of treating osteoarthritis in a subject in need thereof, the method including the step of administering to the subject a first viral vector including a first nucleic acid sequence encoding an sTGF ⁇ -R2 protein or an active fragment thereof; and a second viral vector including a second nucleic acid sequence encoding an ⁇ Klotho protein or an active fragment thereof, thereby treating osteoarthritis in the subject.
• 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.
• the first regulatory sequence drives expression of the sTGF ⁇ -R2 protein or active fragment thereof, and/or the second regulatory sequence drives expression of the ⁇ Klotho protein or active fragment thereof.
• the first regulatory sequence and the second regulatory sequence each comprise a promoter.
• the promoter is a constitutive promoter or an inducible promoter.
• the first regulatory sequence and the second regulatory sequence each comprise a cell-specific promoter or a tissue-specific promoter.
• the first regulatory sequence and the second regulatory sequence each comprise a liver-specific promoter.
• the regulatory sequence comprises a promoter selected from the group consisting of an hEfl ⁇ promoter, an shEfla promoter (or truncated hEfla promoter), a CAG promoter (such as cytomegalovirus, chicken beta-actin intron, splice acceptor of the rabbit beta-globin gene), a CMV promoter, an hAAT promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, a thyroxin-binding globulin (TBG) promoter, a hepatic control region (HCR)- ApoCII hybrid promoter, a C ASI promoter, an HCR- hAAT hybrid promoter, an hAAT promoter combined with mouse album
• the first nucleic acid sequence is operably linked to a 3’ untranslated region for RNA stability and expression in mammalian cells.
• the 3’ untranslated region comprises a sequence selected from the group consisting of a WERE sequence, a WPRE3 sequence, an SV40 late polyadenylation signal (e.g., truncated), an HBG polyadenylation signal, a rabbit beta-globin polyadenylation signal, a bovine bgpA, an ETC polyadenylation signal, and any combination thereof.
• the first viral vector and/or the second viral vector is an adeno-associated virus (AAV) vector.
• the AAV vector is AAV-DJ.
• the AAV vector is derived from an AAV serotype selected from
• AAVl AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV, 11,
• the sTGF ⁇ -R2 protein is selected from the group consisting of a human, a canine, a feline, a bovine, an ovine, a caprine, an equine, a murine, and a porcine sTGF ⁇ R2 protein.
• the sTGF ⁇ -R2 protein is a human sTGF ⁇ -R2 protein.
• the sTGF ⁇ -R2 protein is a canine sTGF ⁇ R2 protein.
• the sTGF ⁇ -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.
• the ⁇ Klotho protein is selected from the group consisting of a human, a canine, a feline, a bovine, an ovine, a caprine, an equine, a murine, and a porcine ⁇ Klotho protein.
• the ⁇ Klotho protein is a human ⁇ Klotho protein.
• the ⁇ Klotho protein is a canine ⁇ Klotho protein.
• the ⁇ Klotho protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:l.
• the sTGF ⁇ -R2 protein and/or the ⁇ Klotho protein is an Fc fusion protein comprising an Ig Fc domain.
• the Ig Fc domain is selected from the group consisting of a human, a canine, a feline, a bovine, an ovine, a caprine, an equine, a murine, and a porcine Fc or a subtype thereof, including IgGl, IgG2a, IgG2b, IgG3, and IgG4.
• the Ig Fc domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID
• the sTGF ⁇ -R2 protein and/or the ⁇ Klotho protein is expressed and distributed systemically.
• the first viral vector and/or the second viral vector is administered by intravenous injection.
• the first viral vector and/or the second viral vector is administered by intra- articular injection into cartilage at osteoarthritic site.
• the first viral vector and/or the second viral vector infects mesenchymal cells at an osteoarthritic site.
• treating osteoarthritis in the subject includes reducing the progression of osteoarthritis in the subject, compared to a control subject.
• osteoarthritis in the subject includes increased, regenerated, or regrown cartilage at an osteoarthritic site in the subject, compared to a control subject.
• treating osteoarthritis in the subject includes reducing inflammation at an osteoarthritic site in the subject, compared to a control subject.
• the subject is a mammal.
• the mammal is a human. According to one aspect, the mammal is a canine.
• Embodiments of the present disclosure are directed to a method of treating osteoarthritis in a subject in need thereof, the method including the step of administering to the subject a therapeutically effective amount of a combination of an ⁇ Klotho protein or an active fragment thereof; and an sTGF ⁇ -R2 protein or an active fragment thereof, thereby treating osteoarthritis in the subject.
• the ⁇ Klotho protein or an active fragment thereof is administered as a soluble protein and the sTGF ⁇ -R2 protein or an active fragment thereof is administered as a soluble protein.
• the ⁇ Klotho protein and/or the sTGF ⁇ -R2 protein is administered by intravenous injection.
• the ⁇ Klotho protein and/or the sTGF ⁇ -R2 protein is administered by intra- articular injection into cartilage at an osteoarthritic site.
• treating osteoarthritis in the subject includes reducing the progression of osteoarthritis in the subject, compared to a control subject.
• treating osteoarthritis in the subject includes increased, regenerated, or regrown cartilage at the osteoarthritic site in the subject, compared to a control subject.
• treating osteoarthritis in the subject includes reducing inflammation at an osteoarthritic site in the subject, compared to a control subject.
• Embodiments of the present disclosure are directed to a method of treating osteoarthritis in a subject in need thereof, the method including the step of administering to the subject a nucleic acid molecule including a first nucleic acid sequence encoding for an ⁇ Klotho protein or an active fragment thereof; and a second nucleic acid sequence encoding for an sTGF ⁇ -R2 protein or an active fragment thereof, thereby treating osteoarthritis in the subject.
• the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element.
• the polycistronic element is an IRES or
• the nucleic acid molecule is administered by intravenous injection.
• the nucleic acid molecule is administered by intra-articular injection into cartilage at an osteoarthritic site.
• treating osteoarthritis in the subject includes reducing the progression of osteoarthritis in the subject, compared to a control subject.
• treating osteoarthritis in the subject includes increased, regenerated, or regrown cartilage at an osteoarthritic site in the subject, compared to a control subject.
• treating osteoarthritis in the subject includes reducing inflammation at an osteoarthritic site in the subject, compared to a control subject.
• the nucleic acid molecule includes DNA, RNA, or a combination thereof.
• the subject is a mammal.
• the mammal is a human.
• the mammal is a canine.
• Embodiments of the present disclosure are directed to a vector including a first nucleic acid sequence encoding an ⁇ Klotho protein or an active fragment thereof; and a second nucleic acid sequence encoding a soluble Transforming Growth Factor Beta Receptor ⁇ (sTGF ⁇ -R2) protein or an active fragment thereof.
• the first nucleic acid sequence and the second nucleic acid sequence are separated by a polycistronic element.
• the polycistronic element is an IRES or 2A sequence.
• a first promoter is operably linked to the first nucleic acid sequence for expression of the ⁇ Klotho protein or an active fragment thereof in a mammalian cell; and a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGF ⁇ -R2 protein or an active fragment thereof in a mammalian cell.
• the first promoter and the second promoter are cell or tissue specific.
• the first promoter and the second promoter are constitutive or inducible.
• the ⁇ Klotho protein and the sTGF ⁇ -R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine and porcine proteins.
• the ⁇ Klotho protein or active fragment thereof and the sTGF ⁇ -R2 protein or active fragment thereof are canine proteins.
• the ⁇ Klotho protein or active fragment thereof and the sTGF ⁇ -R2 protein or active fragment thereof are human proteins.
• the ⁇ Klotho protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:l.
• the sTGF ⁇ -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.
• Embodiments of the present disclosure are directed to a pharmaceutical formulation comprising the vector as described above and a pharmaceutically acceptable excipient.
• Embodiments of the present disclosure are directed to a method of treating osteoarthritis in a mammal in need thereof including the step of administering a therapeutically effective amount of a combination of ⁇ Klotho protein or an active fragment thereof and sTGF ⁇ -R2 protein or an active fragment thereof to the mammal at a site within the mammal exhibiting osteoarthritis, wherein progression of the osteoarthritis is reduced compared to the untreated condition, or wherein cartilage at the site of the osteoarthritis is increased or regenerated or regrown compared to the untreated condition, or wherein inflammation is reduced compared to the untreated condition.
• the mammal is a dog or a human.
• the ⁇ Klotho protein or an active fragment thereof is administered as a soluble protein and the sTGF ⁇ -R2 protein or an active fragment thereof is administered as a soluble protein.
• the ⁇ Klotho protein or an active fragment thereof is administered as a soluble protein by intra- articular cartilage injection and the sTGF ⁇ -R2 protein or an active fragment thereof is administered as a soluble protein by intra- articular cartilage injection.
• a vector comprising a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the sTGF ⁇ -R2 protein or an active fragment thereof.
• a vector comprising a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered by intra- articular cartilage injection and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the sTGF ⁇ -R2 protein or an active fragment thereof.
• a first vector comprising a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second vector comprising a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the sTGF ⁇ -R2 protein or an active fragment thereof.
• a first vector comprising a first nucleic acid sequence encoding the ⁇ Klotho protein or an active fragment thereof and a second vector comprising a second nucleic acid sequence encoding the sTGF ⁇ -R2 protein or an active fragment thereof is administered by intra- articular cartilage injection and the first nucleic acid sequence is expressed to produce the ⁇ Klotho protein or an active fragment thereof and the second nucleic acid sequence is expressed to produce the sTGF ⁇ -R2 protein or an active fragment thereof.
• the vector is a recombinant virus.
• the vector is a parvovirus.
• the vector is an AAV vector.
• the AAV vector is AAV-DJ.
• the vector is an AAV vector serotyped for AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
• the vector infects mesenchymal cells at the site of the osteoarthritis.
• the first vector and the second vector are a recombinant virus.
• the first vector and the second vector are a parvovirus.
• the first vector and the second vector are an AAV vector.
• the first vector and the second vector are an AAV-DJ vector.
• the first vector and the second vector are an AAV vector serotyped for AAV1, AAV2, AAV3, AAV4,
• the first vector and the second vector infect mesenchymal cells at the site of the osteoarthritis.
• the ⁇ Klotho protein or active fragment thereof and the sTGF ⁇ -R2 protein or active fragment thereof are human proteins.
• the ⁇ Klotho protein or active fragment thereof and the sTGF ⁇ -R2 protein or active fragment thereof are canine proteins.
• the ⁇ Klotho protein and the sTGF ⁇ -R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine and porcine proteins.
• the ⁇ Klotho protein has at least 90% sequence identity to the amino acid sequence of an ⁇ Klotho protein corresponding to SEQ ID NO:l.
• the sTGF ⁇ -R2 protein has at least 90% sequence identity to the amino acid sequence of a sTGF ⁇ -R2 protein corresponding to SEQ ID NO:3.
• Embodiments of the present disclosure are directed to a vector including a first nucleic acid sequence encoding an ⁇ Klotho protein or an active fragment thereof and a second nucleic acid sequence encoding a soluble Transforming Growth Factor Beta Receptor ⁇ (sTGF ⁇ -R2) protein or an active fragment thereof.
• a first promoter is operably linked to the first nucleic acid sequence for expression of the ⁇ Klotho protein or an active fragment thereof in a mammalian cell
• a second promoter is operably linked to the second nucleic acid sequence for expression of the sTGF ⁇ -R2 protein or an active fragment thereof in a mammalian cell.
• the first promoter and the second promoter are cell or tissue specific. According to one aspect, the first promoter and the second promoter are constitutive or inducible. According to one aspect, the ⁇ Klotho protein or active fragment thereof and the sTGF ⁇ -R2 protein or active fragment thereof are human proteins. According to one aspect, the ⁇ Klotho protein or active fragment thereof and the sTGF ⁇ -R2 protein or active fragment thereof are canine proteins. According to one aspect, the ⁇ Klotho protein and the sTGF ⁇ -R2 protein are selected from the group consisting of human, canine, feline, bovine, ovine, caprine, equine, murine and porcine proteins. According to one aspect, the ⁇ Klotho protein has at least 90% sequence identity to the amino acid sequence of an ⁇ Klotho protein corresponding to SEQ ID NO:l. According to one aspect, the sTGF ⁇ -R2 protein has at least
• Embodiments of the present disclosure are directed to a pharmaceutical formulation comprising the vector described thereof in a pharmaceutically acceptable excipient.

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• 2020
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