Recombinant AAV vectors expressing bone-protective genes including HAS2 and lubricin useful for treating osteoarthritis and related joint conditions in mammals

Abstract

The present disclosure provides recombinant viral vectors, pharmaceutical compositions comprising such recombinant vectors, and methods for preventing and treating osteoarthritis in mammals. The present disclosure provides adeno-associated virus (AAV) vectors capable of expressing osteoprotective / chondroprotective bioactive proteins, including hyaluronan synthase 2 (HAS2) and lubricin (PRG4), in a host. Methods for producing these AAVs, which are then used to treat osteoarthritis in mammals, are provided through long-term gene expression of the osteoprotective / chondroprotective proteins (including HAS2 and PRG4) in both synoviocytes and chondrocytes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 278,243, filed Jan. 13, 2016, which is incorporated herein by reference in its entirety. SEQUENCE LISTING STATEMENT The sequence listing accompanying this application is provided in text format in lieu of a printout copy and is incorporated herein by reference. The text file containing said sequence listing is named MER 16-291 SEQ Listing_ST25.txt. The text file is 57.6KB and was created on January 13, 2016. It was submitted electronically via EFS-Web contemporaneously with the filing of this application. (Technical field) The present invention relates to recombinant vectors, pharmaceutical compositions comprising such recombinant vectors, and methods for the prevention and / or treatment of acute and / or chronic joint conditions, including osteoarthritis, in animals. In particular, the present invention relates to adeno-associated virus (AAV) vectors capable of expressing in a host biologically active polypeptides belonging to the hyaluronan synthase 2 (HAS2) and lubricin (PRG4) family of proteins. Thus, the present invention relates to the field of genetic engineering, and provides an adeno-associated virus (AAV)-based biological delivery and expression system for use in the treatment of osteoarthritis of human or mammalian joints by long-term gene expression of HAS2 and LUB in synovial cells and chondrocytes. [Background technology]

[0002] Osteoarthritis (OA) is a degenerative joint disease that occurs in mammalian joints and causes serious economic and medical problems (Matthews, GL, and Hunter, DJ (2011). Expert Opin. Emerging Drugs 1-13; Brooks PM. Curr Opin Rheumatol 2002; 14: 573-577). Cartilage is a tough connective tissue that covers the ends of bones at joints. It provides a highly lubricated surface with relatively low friction between hard bones, allowing smooth movement. During the progression of OA, cartilage is partially or completely lost due to abnormal or excessive wear, leading to exposure of the bone ends, which rub against each other, causing inflammation, pain, swelling, or reduced mobility. At present, the exact reasons for the initial cartilage loss that leads to OA are unknown, but there is a strong correlation between its occurrence and age, obesity, and overuse of the joints (e.g., athletic activities). In dogs, osteoarthritis (OA) is one of the most common causes of lameness, affecting an estimated 20 percent of dogs over the age of 1 year. Currently, there is no available treatment that provides a cure for OA, and medical treatment is therefore mostly directed at symptom relief rather than cartilage regeneration. Pain relief treatments usually include steroids and nonsteroidal anti-inflammatory drugs (NSAIDS), which have shown efficacy in treating OA for decades. However, although these drugs can suppress joint inflammation, many of them are known to have degenerative effects on cartilage, which further exacerbates the underlying process of OA progression. In addition to traditional analgesic and anti-inflammatory therapies, direct administration of naturally occurring bone-protecting compounds has been used to alleviate OA symptoms, with varying degrees of success. For example, hyaluronic acid (HA) has been widely used to restore viscoelasticity and lubrication to affected joints. Furthermore, polysulfated glycosaminoglycans (PSGAGs), injected via intra-articular or intramuscular routes, and orally administered glucosamine and chondroitin sulfate have shown some efficacy.

[0003] However, the above mentioned drugs must be administered frequently (sometimes in combination with each other) to achieve meaningful symptom relief. These frequent joint injections are laborious / expensive, pose the risk of infection and cause a great deal of stress to the patient or animal. Surgical approaches have also been developed, but these generally show low efficacy in dogs and horses and are typically only performed in advanced, severely affected subjects. In addition to the delivery of supplements / drugs, several groups have attempted to improve OA symptoms by delivering viscoelastic / viscoprotective polypeptides, nucleic acids encoding same, or polypeptides or nucleic acids capable of being expressed in the host to produce viscoprotective proteins (e.g., enzymes). Approaches of greater interest include the use of lubricin polypeptides (Flannery, US 7,642,236 B2), tribonectins (US 7,618,914 B2 (Rhode Island General Hospital)), and hyaluronan synthases (US 6,423,514 (Millennium Pharmaceuticals)). Some of these efforts can be characterized as "gene therapy" (the basic concepts of which are well established) (Evans CH, Robbins PD. Gene therapy for arthritis, In: Wolff JA (ed.). Gene Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser: Boston, 1994, pp 320-343). Recently, one group has attempted to treat osteoarthritis by in vivo delivery of the interleukin-1 receptor antagonist (Il-1Ra) gene (US 2015 / 0031083 A1 (Baylor College of Medicine); Frisbie, DD et al., Gene Therapy, 2002).

[0004] Arthrogen company has used AAV5 to express human interferon beta (to reduce inflammatory cytokines) in the setting of rheumatoid arthritis (RA). Unlike OA, inflammatory signaling plays a key role in the pathology of RA, therefore blocking this signal is an important therapeutic approach. However, another group pursuing a possible link between inflammation and OA has used recombinant AAV2 to express an IL1 receptor antagonist in the case of equine OA (Goodrich et al., Molecular Therapy-Nucleic Acids (2013) 2, e70). Summary of the Invention [Problem to be solved by the invention]

[0005] However, none of these approaches have proven universally effective, and there remains a significant unmet medical need for relief of pain and suffering in OA patients. As a result, there is a clear and unmet medical need for more effective and durable treatments that are also cost-effective in the long run. Thus, as detailed herein, Applicants have successfully demonstrated for the first time that recombinant adeno-associated virus (rAAV) vectors can deliver cDNA encoding a therapeutic agent by a single intra-articular injection into a mammalian joint, and promote the local and continuous in vivo production of that agent in synovial and chondrocytes. Applicants have also isolated and sequenced, for the first time, the full-length inulbricin cDNA (SEQ ID NO:4). [Means for solving the problem]

[0006] The present invention provides rAAV vectors that express therapeutically effective amounts of bone protective and / or bone regenerative gene products in vivo in a mammalian host. In aspects, the rAAV can include cDNA encoding substances that have disease modifying, lubricating, anti-inflammatory and pain relieving properties.

[0007] In some aspects, the rAAV vector is a vector derived from an AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10. In some embodiments, the AAV nucleic acid comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV2rAAV6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In yet other embodiments, the rAAV particle comprises a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In some embodiments, the ITRs and capsid are derived from the same AAV serotype. In other embodiments, the ITRs and capsid are derived from different AAV serotypes. In some embodiments, the rAAV vector may be of the AAV2 or AAV5 capsid serotype, hi some related embodiments, the rAAV2 and rAAV5 vectors contain at least one ITR from AAV2. In some aspects, the rAAV vector encodes hyaluronan synthase-2 (HAS2) or a variant thereof. In some embodiments, the HAS2 is human HAS2. In other embodiments, the HAS2 is canine HAS2. In some embodiments, the HAS2 is codon-optimized HAS2. The glycosaminoglycan hyaluronanic acid (HA) is a non-sulfated glycosaminoglycan that consists of repeating glucuronic acid and N-acetylglucosamine residues linked by beta-1-3 and beta-1-4 glycosidic bonds. HA serves multiple biological functions, including wound healing, cell migration, malignant transformation, and tissue turnover. HA is synthesized by a variety of cell types, including endothelial cells, fibroblasts, and smooth muscle cells, and has been detected in tissues such as connective, epithelial, and neural tissues. In the joint space, HA is produced by chondrocytes as well as synovial cells, which secrete HA into the synovial fluid. Synovial HA plays a role in joint homeostasis, providing lubrication, tissue hydration, structural integrity, and a scaffold for matrix proteins and biological mechanisms. The biological effects of HA in the joint are determined by its concentration and molecular weight. HA can be synthesized in the range of 5,000 Da to 10,000,000 Da. Smaller molecular weight HA (<500 kDa) is closely associated with receptor-mediated activation of angiogenesis, malignancy and inflammation, whereas higher molecular weight HA provides lubrication in joints.

[0008] Both the size and concentration of joint HA are regulated by its synthesis and degradation rates. Degradation of HA is mediated by hyaluronidases, which cleave HA into smaller fragments that are excreted into the lymphatic system for clearance. Three enzymes, termed hyaluronan synthases (HAS) 1, 2, and 3, have been described for HA production, which are present on the inner surface of synovial cell plasma membranes. Of these, HAS2 has been shown to be required for the production of high molecular weight HA (Itano et al., 1999). High molecular weight HA levels are decreased in osteoarthritis in both human patients and animal OA models (Plickert et al., 2013). This is likely due to decreased HA synthesis and increased HA degradation by hyaluronidases. Of these, HAS2 and 3 are expressed in human cartilage, and of these, HAS2 expression is decreased in human OA. HA levels are also decreased due to increased levels of the HA degrading enzyme hyaluronidase 2 (Yoshida et al.). The HAS2 promoter has been reported to be responsive to a variety of pro- and anti-inflammatory mediators, with opposing effects reported. These mediators include TGFβ, primary epidermal growth factor, TNFα, and retinoic acid (Guo, Kanter el al., 2007; Hyc et al., 2009). Downregulation by inflammatory mediators in diseased joints is expected to reduce HAS2 expression, resulting in reduced HA levels, which may differentially affect the various HAS isoforms (David-Raoudi et al., 2009). In contrast, mechanical stimuli (Momberger et al.2005) or cartilage components (e.g. chondroitin sulfate) have been reported to stimulate HA production (Momberger et al., 2005; David-Raoudi et al., 2009). HA production leads to its presence in the pericellular space as well as its secretion into the extracellular space. It is not clear what regulates the extent of secretion. However, typically, about 80% of hyaluronan is secreted, while the remainder remains associated with the producing cell.This cell-associated hyaluronan is important for the assembly of matrix proteins, and blocking HAS2 synthesis results in a decrease in cell-associated matrix and increased release of proteoglycans (i.e., aggrecan) into the medium, further confirming the primary role of HAS2 as the main enzyme synthesizing hyaluronan in chondrocytes (Nishida et al., 1999).

[0009] In embodiments in aspects, the rAAV vector encodes lubricin or a variant thereof. In some embodiments, the lubricin is human lubricin. In other embodiments, the lubricin is inulbricin. In some embodiments, the lubricin is codon-optimized lubricin. Lubricin (PRG4) is a large mucin glycoprotein produced by synovial lining cells and chondrocytes of cartilage, providing lubrication to protect the cartilage surface (Flannery 1999; Schmidt 2001; Waller 2013). Together with HA, lubricin is also an important lubricant in synovial fluid and provides shock absorbing properties. Lack of lubricin in mouse models and rare genetic diseases in humans results in cartilage degeneration characteristic of osteoarthritis (OA) (Rhee 2005; Ruan 2013). Reduced synthesis of lubricin has also been shown in human OA patients and various animal OA models (Elsaid, 2008). Intra-articular lubricin supplementation with recombinant lubricin has been shown to improve cartilage lesions (Flannery, 2009). In some embodiments, the rAAV vector may be of the AAV2 or AAV5 capsid serotype encoding a canine codon-optimized hyaluronan synthase-2 (HAS2). In some aspects, the rAAV vector is administered by intra-articular delivery. In some embodiments, the rAAV is administered by a single intra-articular delivery. In another embodiment, following intra-articular administration, in vivo production and secretion of cognate therapeutic agent from the rAAV-transduced cells can be sustained for at least about six months. In some embodiments, the rAAV vector comprises an expression cassette comprising a ubiquitous promoter and a codon-optimized species-compatible transgene (see Figure 1A). In another aspect, the disclosure provides methods for expressing bone-protective and / or bone-regenerating gene products in vivo in animal joints using rAAV vectors. All references cited herein, including patent applications and patent publications, are hereby incorporated by reference in their entirety. The following detailed description, provided by way of example and not intended to limit the invention to only the specific embodiments described, will be best understood in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0010] [Figure 1A] FIG. 1A is a schematic diagram showing the HAS expression cassette in a plasmid (top) and in a rAAV viral vector (bottom). [Figure 1B] Figure 1B is a graph showing HA production in cells transfected with cHAS2 expression plasmid. Three days after transfection of 293 cells with cHAS expression plasmid, conditioned medium was harvested and HA levels in the medium were quantified using a HA-binding protein detection system. Abbreviations: CBAcHAS2, HAS2 expression plasmid; EGFP, EGFP expression plasmid; non-transfected, negative control cells; "Optimem" = cells grown in serum-free medium; "Complete", cells grown in serum-containing medium. [Figure 1C] Figure 1C shows an agarose gel image used to separate components of conditioned medium (+ or - hyaluronidase treatment for 24 hours) of cHAS-transfected 293 cells, confirming the expression of HA. Abbreviations: CM, conditioned medium; MDa, molecular weight size of marker. [Figure 2A]Figure 2A is a graph showing rAAV vector production by small-scale packaging of cHAS2 into rAAV2 and AAV5 vectors using a triple transfection method. Packaging of an EGFP expression cassette into AAV2 is shown as a positive control, and packaging of cHAS in the absence of a capsid plasmid is shown as a negative control. rAAV yield is shown as the amount of DNA-resistant particles (DRP) per cell. [Figure 2B] 2B is a graph showing rAAV vector yields in large-scale vector production by triple transfection. Examples of overall titers obtained for multiple vector lots obtained are shown. [Figure 2C] Figure 2C is a graph showing the in vitro performance of AAV2 / HAS2 vectors. 293 cells were infected at various MOIs and HA levels in the conditioned medium were quantified after 3 days. [Figure 2D] Figure 2D is a graph showing the in vitro performance of AAV5 / HAS2 vectors. [Figure 3A] FIG. 3A is a graph showing weight change following intra-articular injection of rAAV / HAS2 vectors in normal dog joints. [Figure 3B] FIG. 3B is a graph showing cartilage scores. [Figure 3C] FIG. 3C is a graph showing synovial scores. [Figure 4A] FIG. 4A is a schematic diagram showing sample collection locations for rAAV vector quantification in canine synovium. [Figure 4B] FIG. 4B is a graph showing quantification of vector genome copy number in synovial sample #3. [Figure 4C] Figure 4C is a graph showing vector genome copy number in synovial sample #1. AAV2 and AAV5 vector doses are indicated as follows: L=low, M=medium and H=high (as in Figures 3A-C). All tissue samples were collected 28 days after rAAV vector delivery and BGHpA was analyzed by qPCR. [Figure 5A]FIG. 5A is a graph showing vector-derived cHAS expression in synovial sample #3. [Figure 5B] FIG. 5B is a graph showing vector mRNA in synovial sample #1. [Figure 5C] Figure 5C is a graph showing the vector genome and vector-derived mRNA for each individual dog analyzed using synovial sample #3. AAV2 and AAV5 vector doses are indicated as follows: L=low, M=medium, and H=high. All tissue samples were collected 28 days after rAAV vector delivery and BGHpA was analyzed by qPCR. [Figure 6A] FIG. 6A is a schematic diagram showing the location of femoral condyle and tibial plateau samples collected for detection of rAAV vector and mRNA in cartilage. [Figure 6B] Figure 6B is a graph showing the vector genome and vector-derived mRNA for each individual dog analyzed using femoral condyle sample #1. In addition, the vector genome copy number for the contralateral (uninjected right joint) is shown (except for sample #22, which was not tested). [Figure 6C] Figure 6C is a graph showing the mean vector genome (injected and non-injected joints) and mRNA copies for each group. AAV2 and AAV5 vector doses are indicated as follows: L=low, M=medium, and H=high. All tissue samples were collected 28 days after rAAV vector delivery and BGHpA was analyzed by qPCR. [Figure 6D] FIG. 6D is a graph showing the average vector genome (PBS or vector injected joints) and mRNA copies in the tibial plateau for each group. [Figure 7A] Figure 7A is a graph showing vector genomes in synovium (samples #3 and #1) and cartilage (femoral condyle and tibial plateau) in each treatment group of tissues collected from the left stifle joint. Values ​​shown in Figures 7A and 7B represent the group mean ± standard deviation (n=5 / group). [Figure 7B]FIG. 7B is a graph showing quantification of vector genome and mRNA derived from the rAAV5 / HAS2 vector in various tissues. [Figure 8A] Figure 8A is a graph showing HA levels in dog synovial fluid. HA levels were quantified in SF samples collected on day -7 (baseline) and day 28. HA levels for each animal were normalized to baseline levels and expressed as % of HA on day 28 versus the week before vector administration. [Figure 8B] Figure 8B is a graph showing HA levels in dog synovial fluid on day -7 (baseline) and day 28. Arrows indicate animals with higher HA levels on day 28 compared to baseline (pre-treatment). [Figure 9] Figure 9 shows the complete amino acid sequence of inulbricin. The boxed region indicates the location of exon 6 (mucin domain). The underlined amino acids (378 to 782) are deleted in the truncated lubricin (hereafter "cLub1" or "cLub1co"), and the location of the KEPAPTT-like repeats (potential O-linked glycosylation sites) are shown in bold. When the sequence name ends with "co", it means that the DNA sequence is codon optimized. Similarly, "nonco" means non-codon optimized. [Figure 10] Figure 10 is a schematic diagram showing the plasmids generated and used in the experiments. The plasmids contain a truncated (i.e., internal deletion engineered), codon-optimized inulbricin sequence (cLub1co), a promoter (minCBA or CBA) and a BGHpA site. Some constructs contain N- or C-terminal His tags (C-term) and intron sequences modified to remove the ATG (potential start codon). The previral AAV lubricin also contains flanking ITR sequences at both ends. [Figure 11A] FIG. 11A is a graph showing the mRNA copies / cell generated upon transfection of minCBA cLub1, CBA CLUB1, and CBH cLub-nonco into 293 cells. [Figure 11B]FIG. 11B is a graph showing the mRNA copies / cell generated upon transfection of ΔATG / 6His / N′, 6His / N′, 6His / C′, WT cLub and EGFP constructs into 293 cells. [Figure 12A] Figure 12A is an anti-lubricin Western blot showing secreted lubricin levels in enrichment media (plasmids as described above). Canine synovial fluid was used as a positive control. [Figure 12B] (B) Western blot showing lubricin production from the previral lubricin expression plasmid. Two clones were analyzed and compared to expression obtained with the minCBA-cLubco plasmid. Media from untransfected cultures and cells transfected with an EGFP expression plasmid were used as negative controls. [Figure 13A] Figure 13A is a graph showing vector yields in small-scale vector production for AAV2 vectors encoding inulbricin. Two cLub clones (- / + 6xHis tag) were analyzed and compared to packaging of previral (ITR-containing) plasmids carrying EGFP and HAS2 expression cassettes. Negative controls included untransfected cells and transfections lacking the AAV2 capsid expression plasmid. [Figure 13B] 13B is a graph showing vector yields in small-scale vector production for AAV5 vectors encoding inulbricin. Previral plasmids for EGFP and cLub expression cassettes were co-transfected with the AAV5 capsid expression plasmid. [Figure 14] Figure 14 is an anti-lubricin Western blot showing in vitro expression of inulbricin derived from rAAV5 vector. Human 293 cells were infected with various amounts of rAAV5 / minCBA-cLub1 for 72 hours, followed by concentration of conditioned media. Media from AAV5 / CBA-EGFP-infected cells was used as a negative control. Media from cells transfected with a previral lubricin expression plasmid was used as a positive control. [Figure 15] FIG. 15 is a table showing a summary of the sequence numbers. [Figure 16-1] FIG. 16 is an alignment of dog and human lubricin. [Figure 16-2] Figure 16-1 continued. [Figure 17] Figure 17 is a graph showing HA levels at various time points in dog synovial fluid using the MMR model. Synovial fluid was collected 1 week before (pre), 2 weeks after OA induction, and before (day 0) and 57, 112, and 182 days after the joint delivery study. [Figure 18A] FIG. 18A is a graph showing rAAV5 vector detection and expression in synovial samples from canine OA joints 182 days after vector administration. [Figure 18B] Figure 18B is a graph showing rAAV5 vector detection and expression in the cartilage (femoral condyle) of a dog OA joint 182 days after vector administration. [Figure 18C] FIG. 18C is a summary of rAAV5 vector genome and cHAS2 mRNA detection in synovial and cartilage samples at day 182 in a canine MMR OA model. [Figure 19] FIG. 19 shows, as an example, Safranin-O stained sections of cartilage surfaces obtained from the medial side of PBS- and two rAAV5 / cHAS2-treated dog joints. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention Osteoarthritis (OA) is one of the most common causes of lameness in mammals and dogs, affecting an estimated 20% of dogs over the age of 1 year. OA is a progressive, degenerative disease that results in pain, inflammation, and reduced joint mobility. New, safe, and effective therapies that improve joint lubrication and reduce inflammation and pain are needed for the management of OA. As disclosed herein, applicants have discovered that recombinant adeno-associated virus (AAV) vectors can be utilized to deliver genes encoding therapeutic agents via a single intra-articular injection, with the goal of providing localized and continuous production of said agents in the joint. rAAV vectors were generated using AAV2 and AAV5 capsid serotypes and encoded canine codon-optimized hyaluronic acid (HA) synthase-2 (HAS2). Twenty-two healthy adult dogs (AAV2 and AAV5 capsid serotype negative) were administered rAAV2 (1.5 and 10x10 11 vg / joint), rAAV5(5x10 11 rAAV5-treated dogs were administered 100 mg / joint (0.01 mg / joint) or PBS (control) via intra-articular injection. No adverse clinical signs were observed during the subsequent 28-day study. Histopathological analysis showed minimal synovial inflammation in rAAV5-treated joints and no significant changes in the rAAV2-treated group. Vector genome (VG) was detected in the synovium of all rAAV-treated joints and in most cartilage samples. rAAV5 vectors resulted in higher VG detection and mRNA expression in both tissues compared to rAAV2. Preliminary analysis also showed a trend toward increased HA levels in the synovial fluid of the joints studied. In summary, our study demonstrated gene transfer to canine joint tissues and an acceptable safety profile by rAAV2 and rAAV5 vectors encoding HAS2 when administered via a single intra-articular injection in a limited number of dogs.

[0012] Canine hyaluronan synthase 2In one aspect of the invention, the disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and a single-stranded DNA genome. The viral capsid of the disclosure can confer uptake of the vector into articular cells with subsequent transport into the cell nucleus, resulting in expression of a therapeutic agent. In some embodiments, the DNA genome comprises one or more AAV inverted terminal repeats (ITRs) flanking one or more expression cassettes for in vivo expression of the therapeutic gene in an animal host. In some embodiments of the invention, no viral genes will be present or expressed from the rAAV genome. In some embodiments of the present invention, once the rAAV is administered to an animal and incorporated into the cells of the animal, the rAAV genome will persist as an extrachromosomal episome. In some embodiments, the rAAV of the present disclosure can persist in articular cells for extended periods of time, such as, but not limited to, greater than about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or 3 years. The episomal rAAV will continue to be expressed, resulting in the production and secretion of therapeutic substances into the synovial fluid, thereby providing local and continuous production of the substances directly to the joint. The selected transgene product can promote joint health by increasing joint lubrication and reducing pain and cartilage degradation.

[0013] In another aspect of the invention, the disclosure provides a method of treating an animal in need thereof, comprising administering to the animal a therapeutically effective amount of a rAAV of the disclosure. In some embodiments, the method comprises administering to a dog the proposed product directly to the affected joint. In some embodiments, a single treatment is sufficient to affect a significant improvement in the animal's condition. In other embodiments, the treatment is repeated. In some embodiments, the treatment is repeated within 2-3 weeks of the first administration, and in other embodiments, the second administration is given more than 3 weeks after the first administration. In some related embodiments, the second dose comprises administration of rAAV with the same therapeutic gene, where the rAAV comprises the same serotype capsid as the first treatment. In other related embodiments, the second dose comprises administration of rAAV with the same therapeutic gene, but where the rAAV comprises a different serotype capsid than the first administration. In some embodiments, the rAAV comprises a serotype 5 capsid. In related embodiments (where repeated administration is desired), the first dose can comprise administration of rAAV with a serotype 5 capsid, and the second dose can comprise administration of rAAV with a serotype 5 capsid. In some aspects, overexpression of HAS2 protein in osteoarthritic joints increases HA levels in synovial fluid and promotes joint health by enhancing the lubricating, anti-inflammatory and pain relieving properties of HA. Overexpression of HAS2 has been shown in vitro to result in increased HA levels in culture medium by a variety of cell types. This has been shown using CHO, 293 and COS cells as either stable or transient transfections. To provide in vivo overexpression of HA in joints, HAS2 cDNA can be delivered to cartilage and / or synovium (the normal site of HA synthesis) by using a rAAV vector encoding a HAS2 expression cassette. Without being bound by any theory, gene transfer into cells would provide the HAS2 expression cassette for sustained expression of HAS2 and subsequent HA production. Because the therapeutic vector would contain a ubiquitous promoter, the vector would not be subject to downregulation by inflammatory mediators present in osteoarthritic joints, unlike the endogenous HAS2 promoter. When the vector is administered by intra-articular injection, it can result in transduction of multiple cell types, the major cell type being synovial cells. Finally, it has been shown that HAS2 alone is sufficient for HA synthesis, and other associated proteins or components appear to be dispensable for HA production in vitro (Yoshida et al.).

[0014] Inulbricin: Multiple features of lubricin production in osteoarthritic joints are increased by intra-articular delivery of recombinant adeno-associated virus (rAAV) vectors encoding lubricin as a potential treatment for osteoarthritis (OA) in dogs. Lubricin is a large secreted glycoprotein that functions as a lubricant and protects the cartilaginous surface of joints. Herein, the discovery and generation of a full-length inulubricin cDNA is described. The cDNA was used to engineer a truncated, codon-optimized version of inulubricin (cLub1co). The latter (truncate, codon-optimized version) was subsequently used to construct various lubricin expression plasmids. These plasmids were characterized for lubricin mRNA and protein production after transfection into HEK293 cells. The data demonstrated the production of both lubricin mRNA and secreted lubricin from each construct. A rAAV vector was generated with a cLub1co expression cassette, demonstrating the feasibility of rAAV / cLub1 vector generation. This synthetic construct was transfected into HEK293 cells, which secreted inulbricin. The methods and compositions described herein can also be used in the therapeutic treatment of osteoarthritis. The terms "treatment" or "therapeutic treatment", as they relate to osteoarthritis, and as they are used herein and in the veterinary field, relate to treatment, support and / or accelerate the treatment of a subject already suffering from or recovering (e.g., in the recovery phase) from osteoarthritis, or treatment that aims to slow and / or reverse cartilage loss in a subject diagnosed with or at risk for osteoarthritis. An important goal of treatment is to reduce the risk of developing cartilage and bone loss. As used herein, a subject is said to suffer from or be at risk for developing osteoarthritis if it is reasonably expected that the subject will suffer from progressive cartilage loss associated with osteoarthritis. Whether or not an individual subject suffers from or is at risk for developing osteoarthritis can be readily determined by a person skilled in the corresponding veterinary or medical field.

[0015] The methods and compositions described herein can also be used for the preventative treatment of osteoarthritis. The terms "prevention," "prophylaxsis," and "preventative treatment," as they relate to osteoarthritis, and as they are used herein and in the fields of human and veterinary medicine, relate to the treatment of healthy subjects or subjects suffering from an unrelated disease, provided that the subject is considered to be at risk for osteoarthritis. Described herein are therapeutic and prophylactic treatments for osteoarthritis that utilize pharmaceutical compositions comprising vectors capable of expressing HAS or lubricin polypeptides in vivo, as well as methods and compositions that induce a sustained increase in hyaluronic acid or lubricin concentrations in joints to reduce or eliminate cartilage loss. As used herein, a composition is said to have "therapeutic efficacy" or be "therapeutically effective" if administration of an amount of the pharmaceutical composition is sufficient to cause a significant improvement in clinical signs or measurable markers of osteoarthritis in a subject mammal suffering from the disease. As used herein, a composition is said to have "prophylactic efficacy" or be "effective" if administration of an amount of the pharmaceutical composition is sufficient to prevent the onset of osteoarthritis in a subject animal. Also described herein are vectors capable of expressing HAS or lubricin, or variants, fragments, or combinations thereof, in a host in vivo. In some embodiments, the HAS or lubricin polypeptides used in the invention are generally compatible with the intended target species (e.g., a vector encoding canine HAS2 is delivered to a dog suffering from OA).

[0016] Exemplary "variants," "derivatives," and the like, as described herein, include, but are not limited to, HAS and lubricin variants, derivatives, and the like, which are encoded by nucleotide sequences that are not strictly identical to the nucleotide sequences disclosed herein, but in which changes in the nucleotide sequence do not change the encoded amino acid sequence, or such changes result in conservative substitutions of amino acid residues, deletions or additions of one or several amino acids, substitutions of amino acid residues with amino acid analogs that do not significantly affect the properties of the encoded polypeptide (e.g., a variant or derivative has greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the desired activity of the wild-type polypeptide), and the like. Examples of conservative amino acid substitutions include glycine / alanine substitutions; valine / isoleucine / leucine substitutions; asparagine / glutamine substitutions; aspartic acid / glutamic acid substitutions; serine / threonine / methionine substitutions; lysine / arginine substitutions; and phenylalanine / tyrosine / tryptophan substitutions. Other types of substitutions, variations, additions, deletions, and derivatives that result in functional HAS or lubricin derivatives are also described herein, and one of skill in the art would readily know how to make, qualify, or select such variants or derivatives, and how to test such variants or derivatives for HAS or lubricin activity. One of skill in the art can optimize expression of the HAS or lubricin polypeptides of the present invention. For example, but not limited to, removing cryptic splice sites, adapting codon usage by introducing a Kozak consensus sequence before the start codon, altering codon usage, or a combination of the above, improves expression.

[0017] A vector for use in the invention can comprise a nucleic acid sequence encoding a canine HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the canine HAS2 polypeptide is a canine HAS2 variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to SEQ ID NO:2. A vector for use in the invention can comprise an inulbricin polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the inulbricin polypeptide is an inulbricin variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to SEQ ID NO:7. Sequence identity or homology can be determined by comparing sequences when aligned to maximize overlap and identity but minimize sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical algorithms. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin & Altschul (Proc. Natl. Acad. Sci. USA 1990, 87, 2264-2268), which has been modified by Karlin & Altschul (Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993, 90, 5873-5877). Another example of a mathematical algorithm used for comparing sequences is the algorithm of Myers & Miller (Myers & Miller, CABIOS 1988,4, 11-17). Such an algorithm has been incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm described by Pearson & Lipman (Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988, 85, 2444-2448).

[0018] Generally, comparison of amino acid sequences can be accomplished by aligning the amino acid sequence of a polypeptide of known structure with that of a polypeptide of unknown structure. The amino acids in the sequences are then compared, and homologous amino acid groups are grouped together. This method detects conserved regions of the polypeptide and reveals amino acid insertions and deletions. Homology between amino acid sequences can be determined by using commercially available algorithms (see also the discussion of homology above). In addition to those specifically mentioned herein, BLAST, Gapped BLAST, BLASRN, BLASTP, and PSI-BLAST programs (provided by the National Center for Biotechnology Information) are also mentioned. These programs are widely used in the art for this purpose and can align the homologous regions of two amino acid sequences. In all of this suite of search programs, gapped alignment routines are essential to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length one is Q=1 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer. The default one-residue penalty (R) for extending a gap is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer. Any combination of values ​​for Q and R can be used to align sequences to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is ​​BLOSUM62, but other amino acid comparison matrices (e.g., PAM) can also be used.

[0019] The terms "protein," "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any length. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, which contain deoxyribonucleotides or ribonucleotides. As used herein, the term "vector" refers to a recombinant DNA or RNA plasmid or virus, which contains a heterologous polynucleotide to be delivered to a target cell, e.g., in vivo. The heterologous polynucleotide can contain a sequence of interest for therapeutic purposes, optionally in the form of an expression cassette. As used herein, a "vector" need not be capable of replication in the ultimate target cell or subject animal. As used herein, the term "recombinant" means a polynucleotide of semisynthetic or synthetic origin, which is not naturally occurring or which is linked to another polynucleotide in an arrangement not found in nature. The term "heterologous" as used herein means derived from an entity that is genetically distinct from the rest of the entity to which it is being compared. For example, a polynucleotide can be placed by genetic engineering into a plasmid or vector derived from a different source, and thus the polynucleotide is a heterologous polynucleotide. A promoter that has been removed from its native coding sequence and operably linked to a coding sequence other than its native sequence is thus a heterologous promoter. Polynucleotides used in accordance with the invention can include additional sequences such as, for example, additional coding sequences within the same transcription unit, control elements (e.g., promoters), ribosome binding sites, transcription termination factors, polyadenylation sites, additional transcription units under the control of the same or different promoters, sequences allowing for cloning, expression, homologous recombination in host cells, and any other constructs that may be desired to provide an embodiment of the invention.

[0020] In one aspect, the disclosure provides a method of treating a subject mammal suffering from or at risk for osteoarthritis, comprising administering to the subject mammal a therapeutically effective amount of an adeno-associated virus (AAV) comprising a nucleic acid sequence encoding an osteoprotective or bone regenerating polypeptide, optionally operably linked to a promoter, wherein the polypeptide is expressed in vivo in the subject mammal in an amount effective to alleviate or prevent a symptom of OA. In some embodiments, administration is by an intra-articular route. In some embodiments, the polypeptide can encode hyaluronic acid synthase (HAS) (including HAS2), lubricin, interleukin-1 receptor (IL-1R) antagonist, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2), transforming growth factor beta 1 (TGFβ1), bone morphogenetic protein 7 (BMP7), glucosamine-fructose-6-phosphate aminotransferase (GFAT), interleukin 10 (IL-10), heme oxygenase-1 (HO-1), biologically active truncated forms thereof, or combinations thereof. In some embodiments, the subject mammal can be a human, a dog, or a cat. In a specific embodiment, the subject mammal is a dog. In some embodiments, the subject mammal is suffering from or at risk of developing chronic osteoarthritis.

[0021] In other embodiments, the polypeptide is canine HAS2 or inulbricin. In some embodiments, a nucleic acid sequence encoding a HAS2 polypeptide has a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:3, or a nucleic acid sequence encoding a lubricin polypeptide has a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:6. In some embodiments, the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the HAS2 polypeptide has an amino acid sequence selected from polypeptides having at least 90% identity to the sequence set forth in SEQ ID NO: 2, fragments, variants, and homologs thereof, each of which exhibit in vivo HAS activity in a subject animal. "HAS activity" refers to the production of biologically active hyaluronan. In some embodiments, the AAV vector comprises the following elements from 5' to 3': 5' AAV ITR, stuffer, CBA, intron (IN), cHAS2 codon-optimized cDNA, polyadenylation signal (pA), and 3' AAV ITR. In some embodiments, the lubricin polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7. In other embodiments, the lubricin polypeptide has an amino acid sequence selected from polypeptides having at least 90% identity to the sequence set forth in SEQ ID NO:7, fragments, variants, and homologs thereof, each of which exhibit in vivo lubricin activity in a subject animal. "Lubricin activity" means providing lubrication in substantially the same manner and to substantially the same extent as endogenously produced lubricin. Such lubricity activity can be measured according to techniques known in the art (see, e.g., Swan, DA et al. Biochem J. 1985 Jan 1; 225(1): 195-201). In some embodiments, the promoter can be selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, an adenovirus major late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase (CK) gene promoter. In yet other embodiments, the AAV comprises an AAV2 or AAV5 capsid.

[0022] In another aspect, the disclosure provides a method for increasing the production of hyaluronic acid in both chondrocytes and / or synovial cells of a mammal (e.g., a human or a canine). In one embodiment, the method includes administering to the mammal (e.g., a human or a canine) a recombinant AAV ("rAAV") comprising a rAAV vector genome (including a nucleic acid encoding HAS2), allowing sufficient time for the HAS2 enzyme to be expressed and thereafter catalyze the production of additional hyaluronic acid, thereby increasing the hyaluronic acid levels in the mammal. The disclosure also provides methods for increasing production of lubricin polypeptide in both chondrocytes and / or synovial cells of a mammal (e.g., a human or a canine). In certain embodiments, the methods include administering to the mammal (e.g., a human or a canine) an rAAV that includes an rAAV vector genome (including a nucleic acid encoding lubricin), allowing sufficient time for lubricin to be expressed, thereby increasing lubricin levels in the canine. In one embodiment, HAS2 is produced in sufficient amounts following administration of a rAAV comprising a nucleic acid encoding HAS2 to treat or prevent symptoms of OA in a mammal (e.g., a human or a dog). In another embodiment, lubricin is produced in sufficient quantities following administration of an rAAV containing a nucleic acid encoding lubricin to treat or prevent symptoms of OA in a mammal (e.g., a human or a dog). In one embodiment, HA levels are restored to levels found in a healthy mammal (e.g., a human or a dog). Those skilled in the art can consult various references to learn what HA levels are found in healthy animals (e.g., Smith, GN et al. Arthritis Rheum. 1998; 41:976-985; Balazs E et al. Disorders of the Knee. Philadelphia: JB Lippincott; 1982. pp. 61-74). In another embodiment, lubricin levels are restored to levels found in a healthy mammal (e.g., a human or a dog).

[0023] In another aspect, the disclosure provides a method of treating a dog suffering from or at risk of developing OA, the method comprising administering to the dog a therapeutically effective amount of an AAV vector comprising a nucleic acid sequence encoding a HAS2 or lubricin polypeptide operably linked to a promoter. In another embodiment, the disclosure provides a method of treating a human suffering from or at risk of developing OA, the method comprising administering to the human a therapeutically effective amount of an AAV vector comprising a nucleic acid sequence encoding a HAS2 or lubricin polypeptide operably linked to a promoter. In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO:3, or the nucleic acid sequence encoding the lubricin polypeptide has at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO:6. In some embodiments, the AAV encodes a HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the AAV encodes a lubricin polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7, or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7. In any embodiment, the promoter can be selected from a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, an adenovirus major late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a beta-actin gene promoter, a CBA promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter.

[0024] In another aspect, the disclosure provides a method of preventing the development of OA in a subject mammal at risk thereof, comprising administering to said dog a therapeutically effective amount of an rAAV comprising an rAAV vector genome comprising a nucleic acid sequence encoding a HAS2 or lubricin polypeptide operably linked to a promoter. In one embodiment, the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO:3, or the nucleic acid sequence encoding the lubricin polypeptide has at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO:6. In another embodiment, the nucleic acid encodes a HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or the nucleic acid encodes a lubricin polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, the promoter can be selected from the group consisting of a CMV IE promoter, a RSV promoter, a HSV-1 TK promoter, a SV40 early promoter, a SV40 late promoter, an adenovirus major late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter. In some embodiments, the rAAV vector comprises CBA-cHAS2co-BGH. In other embodiments, the rAAV vector comprises pITR / minCBA-HIb-cLub1co-BGH.

[0025] In another embodiment, the disclosure provides a recombinant plasmid vector comprising a nucleic acid sequence encoding a canine HAS2 or lubricin polypeptide operably linked to a promoter. In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO:3, or the nucleic acid sequence encoding the lubricin polypeptide has at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO:6. In some embodiments, the nucleic acid encodes a HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or the nucleic acid encodes a lubricin polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7. In one aspect, the disclosure provides a pharmaceutical composition comprising a recombinant viral vector encoding and expressing HAS or lubricin in vivo in a mammalian host, and optionally one or more pharma- ceutical acceptable carriers, excipients, or vehicles. In another embodiment, the present disclosure provides a method of treating a mammalian subject suffering from or at risk of developing osteoarthritis, said method comprising the step of intra-articularly administering to said mammalian subject a therapeutically effective amount of a pharmaceutical composition as detailed above. In one embodiment, the subject is a human or a dog. In another aspect, the disclosure provides an adeno-associated virus (AAV)-based biological delivery and expression system for use in the treatment or prevention of OA of human or mammalian joints. In some embodiments, the method is achieved by long-term gene expression of human or mammalian HAS2 or lubricin in synoviocytes and / or chondrocytes followed by delivery of rAAV, which comprises a nucleic acid sequence encoding human or mammalian HAS2 or lubricin, left and right AAV inverted terminal repeats (L ITR and R ITR), an AAV packaging signal, and optionally a non-viral non-coding stuffer nucleic acid sequence. In some embodiments, expression of the human or mammalian HAS2 or lubricin gene in synoviocytes and / or chondrocytes is regulated by an inflammation-inducible promoter. The promoter is placed upstream of the reading frame of the nucleic acid sequence encoding human or mammalian HAS2 or lubricin and is specifically activated by increasing levels of an immune stimulant.

[0026] In some embodiments, the inflammation-inducible promoter is selected from the following: NF-KB promoter, interleukin 6 (IL-6) promoter, interleukin-1 (IL-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1a (MIP-1a) promoter, and hybrid constructs thereof. In some embodiments, the rAAV vector genome comprises a nucleic acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:6, or a biologically effective variant thereof. In some embodiments, the AAV system comprises a nucleic acid encoding a marker gene that allows monitoring of the vector genome in synovial cells and chondrocytes. In some embodiments, the vector comprises a nucleic acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:6, or a conserved sequence thereof encoding the same amino acid. In some embodiments, the rAAV vector genome comprises a nucleic acid encoding a HAS2 polypeptide as set forth in SEQ ID NO:2 or a lubricin polypeptide as set forth in SEQ ID NO:7. The rAAV vector genome comprises a nucleic acid molecule having at least 80% or 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3. In other embodiments, the rAAV vector genome comprises a nucleic acid molecule having at least 80% or 90% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6. In some embodiments of the AAV system, the system comprises a nucleic acid sequence encoding human or mammalian HAS2 or lubricin, left and right AAV inverted terminal repeats (L ITR and R ITR), a packaging signal, and optionally a non-viral non-coding stuffer nucleic acid sequence, and expression of the human or mammalian HAS2 or lubricin gene in synoviocytes and / or chondrocytes is regulated by an inflammation-inducible promoter and specifically activated by increased levels of an immune stimulant for the treatment or prevention of osteoarthritis (OA).

[0027] Viral particles and methods for producing viral particlesAlso provided herein is a viral particle comprising a nucleic acid encoding HAS2 or lubricin. A viral vector can be used to deliver a nucleic acid encoding HAS2 or lubricin to express the protein in target cells at a specific location (e.g., a joint). Many types of viruses are known, and many have been studied for the purpose of delivering nucleic acids to target cells. Exogenous nucleic acids can be inserted into a vector (e.g., adeno-associated virus (AAV)). In some embodiments, the viral particle is a recombinant AAV particle that includes a nucleic acid comprising one or two AAV ITRs and a sequence encoding HAS2 or lubricin as described herein flanked by one or two ITRs. The nucleic acid is encapsulated within the AAV particle. The AAV particle also includes a capsid protein. In some embodiments, the nucleic acid includes operably linked transcriptional components, control sequences (including transcriptional start and stop sequences), and a protein coding sequence of interest (e.g., a nucleic acid encoding a fusion protein). These components are flanked at the 5' and 3' ends by functional AAV ITR sequences. By "functional AAV ITRs" it is meant that the ITRs function as intended for rescue, replication and packaging of AAV virions (see Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J.Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated by reference in their entireties). To carry out some features of the invention, the recombinant vector contains at least all of the sequences of AAV essential for physical structure for encapsulation and infection by rAAV. The AAV ITRs used in the vectors of the invention need not have wild-type nucleotide sequences (e.g., as described by Kotin (Kotin, Hum. Gene Ther., 1994, 5:793-801)) but may be modified by nucleotide insertions, deletions or substitutions or may be derived from any of several AAV serotypes. To date, over 40 AAV serotypes are known, and new serotypes and variants of existing serotypes continue to be identified. See, e.g., Gao et al., PNAS, 2002, 99(18): 11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is considered within the scope of the invention.In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10. In some embodiments, the AAV nucleic acid comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In yet other embodiments, the rAAV particle comprises a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10.

[0028] Different AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types within a particular target (e.g., joints). rAAV particles can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, rAAV particles can contain AAV2 capsid protein and at least one AAV2 ITR, or they can contain AAV2 capsid protein and at least one AAV5 ITR. In another example, rAAV particles can contain AAV5 capsid protein and at least one AAV2 ITR. Any combination of AAV serotypes is provided herein for the production of rAAV particles as if each combination were expressly described herein. rAAV particles can be produced using methods known in the art. See, e.g., U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006. In the practice of the invention, host cells for producing rAAV particles include mammalian cells, insect cells, plant cells, microorganisms, and yeast. The host cell can also be a packaging cell, in which the AAV rep and cap genes can be stably maintained in the host cell or in the producer cell in which the AAV vector genome is stably maintained. Exemplary packaging and producer cells are derived from 293, A549, or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

[0029] In some aspects, methods of producing any of the rAAV particles disclosed herein are provided, comprising: culturing a host cell under conditions whereby rAAV particles are produced, wherein the host cell comprises (i) one or more AAV packaging genes, each encoding an AAV replication or encapsulation protein, (ii) a rAAV provector comprising a nucleic acid encoding any of the fusion proteins disclosed herein flanked by at least one AAV ITR, and (iii) AAV helper functions; and (b) recovering the rAAV particles produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10 ITRs. In some embodiments, the encapsulation protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10 capsid proteins. In yet another embodiment, the rAAV particles are purified. As used herein, the term "purified" includes rAAV particle preparations that lack at least some of the other components that may also be present in the location where the rAAV particles naturally occur or where the rAAV particles were originally prepared. Thus, for example, isolated rAAV particles can be prepared using purification techniques that enrich the particles from a source mixture (e.g., culture lysate or production culture supernatant). Enrichment can be measured in a variety of ways, such as by the percentage of DNase-resistant particles (DRP) present in solution or by infectivity. This can also be measured relative to a second potentially interfering substance (e.g., a contaminant) present in the source mixture. The above-mentioned contaminants include contaminants from the production culture or contaminants during processing (including helper viruses, medium components, etc.). Also provided herein is a pharmaceutical composition comprising rAAV particles comprising a nucleic acid encoding HAS2 or lubricin of the invention and a pharma- ceutically acceptable carrier. The pharmaceutical composition may be suitable for a variety of modes of administration (including, for example, systemic or local administration) as described herein. The pharmaceutical composition of rAAV comprising a nucleic acid encoding HAS2 or lubricin as described herein may be introduced systemically, for example, by intravenous injection, by catheter (see U.S. Pat. No. 5,328,470), or by stereotactic injection (Chen et al., 1994, PNAS, 91: 3054-3057). In some embodiments, the composition comprising the rAAV described herein and a pharma- ceutically acceptable carrier is suitable for administration to humans. Such pharma- ceutical acceptable carriers may be sterile liquids, such as water and oils (including oils of petroleum, animal, vegetable, or synthetic origin), such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions can further include additional ingredients, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, thickening agents, and the like. The pharmaceutical compositions described herein can be packaged in single unit dosage forms or in multi-unit dosage forms. The compositions are generally formulated as sterile, substantially isotonic solutions.

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Phase 1 dose-escalation safety and tolerability study.Ann Rheum Dis 68:1247-1254 Mietzsch M et al.2014.Differential adeno-associated virus serotype-specific interaction patterns with synthetic heparins and other glycans.J Virology 88:2992-3003 Miltner O et al.Efficacy of intraarticular hyaluronic acid in patients with osteoarthritis-a prospective clinical study.Osteoarthritis Cartilage 2002, 10:680-6Mingozzi F et al.2013.Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue.Gene Therapy 20:417-424 Momberger TS et al.Hyaluronan secretion by synoviocytes is mechanosensitive.Matrix Biology 2005, 24:510-519 Nishida Y et al.Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly.JBC 1999, 274:1893-21899 Ortved KF et al.Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model.Mol Ther 2015, 23:363-373 Payne KA et al.Single intra-articular injection of adeno-associated virus results in stable and controllable in vivo transgene expression in normal rat knees.Osteoarthritis Cartilage 2011, 19:1058-1065 Plickert HD et al.Hyaluronic acid concentrations in synovial fluid of dogs with different stages of osteoarthritis.Research in Veterinary Science 2013, 94:728-734 Rapti K et al.2012.Neutralizing antibodies against AAV serotypes 1, 2, 6, and 9 in sera of commonly used animal models.Molecular Therapy 20:73-83 Rhee DK et al.Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing.JBC 2005, 280:3125-3132 Ryan MZC et al.Proteoglycan 4 expression protects against the development of osteoarthritis.Sci Transl Med 2013, 5:176ra34 Sarzi-Puttini P et al.Osteoarthritis: an overview of the disease and its treatment strategies.Semin.Arthritis Rheum 2005, 35:1-10 Schmid T et al.2001.Superficial zone protein (SZP) is an abundant glycoprotein in human synovial fluid and serum.Trans Orthop Res Soc 26:82 Sharkey M.The challenges of assessing osteoarthritis and postoperative pain in dogs.2013.The AAPS Journal 15:598-607 Vugmeyster Y et al.Disposition of human recombinant lubricin in naive rats and in a rat model of post-traumatic arthritis after intra-articular or intravenous administration.AAPS J 2012, 14:97-104 Waller KA et al.Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis.PNAS 2013 Watanabe K and Yamaguchi Y.Molecular identification of a putative human hyaluronan synthase.JBC 1996, 271:22945-48 Watanabe, S et al.Adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective IL-4 to murine synovium.Molecular Ther 2000; 2: 147-151 Watson RS et al.scAAV-mediated gene transfer of interleukin-1-receptor antagonist to synovium and articular cartilage in large mammalian joints.Gene Therapy 20:670-677 Yoshida M et al.Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction.Arthritis Research Therapy 2004, 6:R514-R520 EXAMPLES

[0031] Construction and evaluation of HAS2 AAV vectors Example 1a - Overview rAAV vectors containing codon-optimized canine HAS2 cDNA and a ubiquitous promoter were generated and packaged into AAV2 or AAV5 capsids. Large-scale vector lots were generated by triple transfection and purified by CsCl gradient. Vector yields were quantified by qPCR against the bovine growth hormone (BGH) polyA site (pA). A total of four lots were generated for AAV2 / HAS2 (three of which were pooled for in vivo testing, 2x10 13 DRP / total amount. For AAV5 / HAS2, there are two lots (5x10 12 The consistency of the production yield was tested to obtain sufficient amounts of virus. Twenty-two healthy adult dogs (AAV2 and AAV5 capsid serotype negative) were administered rAAV2 (1, 5 and 10x10 11 vg / joint), rAAV5(5x1011 rAAV5 treated joints were administered 100 mg / vg / joint, or PBS (control) via intra-articular injection. Histopathological analysis showed minimal synovial inflammation in rAAV5 treated joints and no significant changes in the rAAV2 treated group. Vector genome (VG) was detected in the synovium of all rAAV treated joints and in the majority of cartilage samples. rAAV5 vectors resulted in higher VG detection and mRNA expression in both tissues compared to rAAV2. A trend towards increased HA levels in the synovial fluid of treated joints was observed. In summary, the disclosed results demonstrated gene transfer to canine joint tissues and an acceptable safety profile when administered via a single intra-articular injection in dogs.

[0032] Example 1b - Method Cloning and construction of HA expression vector The canine HAS2 gene (GenBank XM 539153.3 (SEQ ID NO: 1)) was codon-optimized for expression in canine by the GeneArt / Invitrogen algorithm. The codon-optimized canine HAS2 cDNA (1656 bp (SEQ ID NO: 3)) was synthesized with NheI-NsiI restriction enzyme sites flanking it. This fragment was then cloned into a plasmid containing the ubiquitous chicken b-actin promoter (CBA), a hybrid intron and bovine growth hormone (BGH) polyA (pA). The resulting pCBA-HI-cHAS2-BGHpA plasmid was purified for expression analysis using a maxi kit (Qiagen). In vitro expression of cHAS2 and HA production: Plasmid vectors containing cHAS2 were transfected into 293 cells, and conditioned media and cell lysates were collected after 3 days in 250 μL of RIPA buffer + protease inhibitors. Cell lysates were spun to remove cell debris, and 30 μL of cell lysate was loaded onto a 4-12% nu-PAGE gel and run in 1x Mops buffer. The protein gel was transferred to a nitrocellulose membrane and probed with anti-HAS2 in 5% milk and 0.1% Tween-20 in PBS-T overnight at 4°C. Donkey anti-goat secondary antibody (1:5000 dilution) was used as the secondary antibody. Beta-actin detection was used to demonstrate equal loading of cell lysates. Quantification of hyaluronan levels and molecular weight in in vitro cultures : HA production by HAS2 expressing cells was assessed by transfecting 293 cells with pCBA-HI-cHAS2-BGHpA (with Optimen or complete medium). HA levels in conditioned media were quantified using an HA test kit (Corgenix, Inc.), which contains an HA-binding protein derived from aggrecan. The molecular weight of HA was assessed by running concentrated conditioned media on an agarose gel. Various HA size markers were run in parallel (Select-HA HiLadder, Hyalose, Austin, TX). Similar gels were run in parallel and subsequently digested with hyaluronidase for 24 hours. Both gels were stained with All-stain. Construction of rAAV vectors carrying cHAS The cHAS2 expression cassette was cloned into an AAV ITR-containing plasmid to generate an expression cassette flanked by AAV inverted terminal repeats (previral plasmid pDC627), constructing psITR / CBA-HI-cHAS2-BGHpA. 600 bp of stuffer DNA (chromosome 16 P1 clone 96.4B) was added upstream of the expression cassette, generating a total viral vector genome of 4500 bp. To test packaging of the cHAS2 expression cassette-containing plasmid, 293 cells were cultured at 8x10 5Cells / well were seeded (6-well plates) and transfected the next day in duplicate with psITR / CBA-HI-cHAS2-BGHpA or psp70 / EGFP, pHLP-19cap2 or p5repCMVcap5 plasmids and pAdHELP (CaPO4 kit (Promega)). After 3 days, cells were harvested and lysates were titered for BGHpA copies using qPCR and primers / probes of the BGHpA sequence (SEQ ID NO:12-14). Plasmids containing BGHpA were used as standards. rAAV virus yields were expressed as the amount of DNase-resistant particles (DRP) per cell. Large-scale vector production was performed using triple transfections of psITR / CBA-HI-cHAS2-BGH, pIM45BD rep-cap plasmid for AAV2 vectors, and pHLP19-cap5 and pAdHELP for AAV5 vectors. Vectors were purified by CsCl and the resulting vector lots were titered using TaqMan analysis and a primer / probe for the BGHpA sequence (Applied Biosystems / Life Technologies). In vitro efficacy of rAAV / cHAS2 in rabbit chondrocytes and synovial cells The ability of the vector to transduce articular cell types (e.g., primary synoviocytes and chondrocytes) was tested in rabbit cells. Cells were infected with 1e5 DRP / cell and cultured for 3 days. Cell lysates were collected to detect HAS2 protein by Western blot, and HA levels were quantified as described above. To test the effect of HA on the production of matrix-degrading proteases, inflammatory cytokines, and cartilage structural proteins under symptoms, cells were first infected with rAAV vectors, followed by IL-1b stimulation 24 hours later. After 24 hours, both cells and culture medium were collected for mRNA analysis and HA production. Evaluation of rAAV / cHAS2 in normal canine joints Mongrel dogs (male and female, 8-10 kg / kg) were used. Dogs with serum titers of 4 or less for AAV2 and / or AAV5 capsids were used in the study. rAAV and rAAV5 vectors encoding cHAS2 were administered via the intra-articular route (AAV2: 1, 5 and 10x10 11DRP / Joint, AAV5:5x10 11 DRP / joint). PBS was used as a negative control. Animals were observed for clinical signs once daily for 7 days before injection, twice daily for 7 days after injection, and once daily thereafter for the duration of the study (pain, crawling, swelling of the injected joint, and other abnormalities). Animals were sacrificed after 4 weeks. Whole blood samples were collected on days -7, 1, 14, and 28 after vector administration for white blood cell (WBC) counts. Synovial fluid (SF) samples were collected on days -7, 1, 14, and 28 for quantification of HA levels. Synovial tissue, cartilage, and liver samples were collected for DNA and RNA isolation. cHAS2 vector genome and mRNA copies were determined by qPCR analysis using the BGHpA primer / probe set (Applied Biosystems / Life Technologies). Sections were stained with toluidine blue and reviewed by a board-certified veterinary pathologist. Cartilage was assessed for severity of cartilage lesions and proteoglycan loss (score 0-5). Since no synovial thickening was observed, the synovial lesion score was determined by the density of inflammatory cells (score 0-5).

[0033] Example 1c - Results Codon optimization and construction of HAS2 expression cassetteMammalian HAS2 is a highly conserved protein. For example, the human and canine amino acid sequences of HAS2 differ by only two amino acids (99.3% identity). Similarly, there are only three amino acid differences between canine and rabbit HAS2 (99.5%). At the DNA level, the similarity between canine and human HAS2 synthase cDNA is 93.9%. Since gene expression can be improved by codon optimization, the GenBank sequence of canine HAS2 (XM 539153.3) was optimized by GeneArt / Invitrogen. This resulted in a nucleotide sequence with 78% similarity to the original GenBank sequence. The GC content of the optimized cDNA was increased from 44.4% to 59.0%. This cDNA was used to generate a ubiquitous expression plasmid with a CBA promoter, allowing constitutive expression of HAS2 distinct from the endogenous promoter (Figure 1A). The CBA promoter is less affected by a variety of pro- and anti-inflammatory cytokines. HAS2 expression and HA production in vitroTo test the in vitro expression of HAS2 protein, two clones (#1 and #2) of the CBA-HI-cHAS2-BGHpA plasmid vector were transfected into 293 cells, and the cell lysates were subsequently analyzed for HAS2 protein (membrane protein) by Western blot. Cells transfected with the expression plasmid showed a band of 64 kDa, the expected size of cHAS (data not shown). We next determined whether overexpression of HAS2 protein in 293 cells resulted in increased detection of HA in the culture medium (indicating both the production of HA and the secretion of HA across the cell membrane). HA levels in the medium of cells transfected with pCBA-cHAS2 were increased 6.5- and 9-fold compared to non-transfected and CBA-EGFP-transfected cells, respectively (Figure 1B). Thus, the data confirmed that overexpression in cells results in increased levels of HA in the extracellular compartment. The size of HA produced in vitro was determined by agarose gel. The data demonstrated the presence of high molecular weight HA in conditioned medium from cells transfected with the HAS2 expression cassette. The size of this material was greater than 1.5 megadaltons (MDa) (based on estimates from HA molecular weight markers). This material disappeared after hyaluronidase digestion, indicating that the material was HA (Figure 1C). Construction of rAAV vector carrying a HAS2 expression cassette The cHAS2 co-expression cassette was then cloned into a plasmid with AAV ITRs. A schematic of the resulting viral genome is shown in Figure 1A. The ability to generate rAAV vectors with AAV2 and AAV5 capsids and HAS2 cDNA was tested in small-scale packaging experiments (Figure 2A), followed by large-scale vector production. Using a standard triple transfection method, both AAV2 and AAV5 vectors could be generated (Figure 2B). The potency of this material was tested by infection of 293 cells and analysis of the levels of HA produced in the culture medium. Both AAV2 and AAV5 vectors produced a dose-response increase in HA in the culture medium (Figure 2C,D). Evaluation of rAAV / HAS2 vectors in normal canine jointsrAAV2 and rAAV5 vectors carrying cHAS2 were delivered by intra-articular injection into normal dog joints and animals were evaluated for 28 days. No adverse clinical signs, weight changes (Figure 3A), crawling or death were observed during the study. White blood cell (WBC) counts were elevated in some animals on day -7, possibly due to the stress of transportation. WBC counts on days 1, 14 and 28 were generally within normal limits. Histological evaluation of PBS-, AAV-injected (left) and contralateral (non-injected) knees showed very minimal proteoglycan loss and cartilage degeneration (score range 0-0.5 (maximum score 5)). These minimal changes were typical of naturally occurring changes associated with age. Minimal synovial changes were observed in PBS- and AAV2-treated joints as well as in the contralateral joint (Figure 3C). Minimal to mild synovitis (generally involving the joint capsule and medial collateral ligament) was noted in all left knees of males and females treated with AAV5 vectors (no synovitis was observed in the contralateral joints), therefore, overall the treatment was well tolerated with few observed adverse effects. Tissue samples collected from synovium and cartilage were analyzed for detection of viral genomes (Fig. 4A, 6A). Synovial samples collected from the site closest to the injection (sample #3) showed the presence of vector genomes in all of the AAV-treated joints (Fig. 4B). AAV2-treated joints contained roughly 0.01 to 2 vector genomes (VG) / cell. Interestingly, a minor dose response was observed with AAV2, despite a 10-fold difference between the low and high dose groups. Joints treated with AAV5 vectors showed higher and more consistent detection, ranging from 1 to 12 copies / cell. Low levels of VG were detected in some contralateral (non-injected) joints, which were more evident in the low AAV2 treatment group and more sporadic in the high AAV2 dose and AAV5 groups (data not shown). A synovial sample collected further above the injection site (sample #1) was analyzed to assess AAV spread within the joint (Figure 4C). Joints injected with low dose AAV2 showed more consistent VG detection. These levels are similar to those measured in synovial sample #3. In the AAV5 treatment group, all synovial #1 samples had consistently detectable VG (within 3-fold); however, these were lower than the VG levels detected in synovial #3, thus revealing location-dependent transduction. Expression from the vector genome was analyzed by quantification of vector-derived mRNA. For synovial sample #3, expression was detected in 2 / 5, 4 / 5, and 4 / 5 of the AAV2-treated low, medium, and high dose groups, while all AAV5-treated joints had detectable mRNA copies (Figure 5A). Vector expression was also detected in synovial #1 for the AAV5 vector, but the levels were low, similar to the low VG detection at this location (Figure 5B). The detection of mRNA correlated well with the VG detection. The mRNA and VG DNA of each of the individual injected joints of synovial sample #3 are shown as examples (Figure 5C).

[0034] Detection of vector genome in canine cartilage Cartilage samples collected from the femoral condyle and tibial plateau were analyzed for detection of viral genomes (VG, Fig. 6A). Vector DNA and mRNA detected in each individual injected joint and group average in the femoral condyle are shown as examples (Fig. 6B, C). The data show that AAV5 vectors are present in a consistent manner in cartilage and further showed comparable levels of vector-derived transcripts. A similar dose of AAV2 vector (medium dose) produced similar VG levels as AAV5 vectors but 100-fold lower mRNA levels. In addition, AAV2 VG copies appeared to be inversely correlated with vector dose. rAAV5 injected joints also showed vector detection and expression in cartilage samples collected from the tibial plateau, whereas none were detected in rAAV2 treated joints (Fig. 6D). All vectors produced minimal vector DNA detection in the contralateral (non-injected) joint. The synovium and cartilage results are summarized in Figure 7A. For gene transfer in synovium, AAV5 vectors yielded approximately 10-fold higher vector copies in both synovium sample locations compared to AAV2 vectors. Gene transfer into cartilage was 10-20-fold lower than that into synovium by AAV5, whereas AAV2 vector genomes were observed at similar levels in both synovium and cartilage. Detection of rAAV5 vector-derived genomes and mRNA is summarized in Figure 7B, showing consistent gene transfer and expression by rAAV5 / HAS2 vectors in all tissue samples tested. Applicants believe this result to be highly unexpected. Analysis of synovial fluid HA levels To determine whether any changes in HA levels in synovial fluid could be detected following rAAV vector administration, synovial HA levels were quantified in samples collected on day -7 (baseline) and day 28. Because a high level of variation was detected between animals, HA levels for each animal were normalized to each animal's baseline level. The data showed that both AAV2 / high dose and AAV5 / medium dose increased HA levels in synovial fluid on average compared to PBS-treated animals (Figures 8A and 8B).

[0035] Example 1d - Conclusion To provide in vivo overexpression of HA in joints, applicants generated rAAV vectors with two capsid serotypes. The choice of AAV capsid serotype is important because any neutralizing antibodies already present in the target species may neutralize the therapeutic vector and therefore block gene transfer by the rAAV vector. The results disclosed herein showed that the majority of dogs analyzed had low levels of neutralizing antibodies to both AAV2 and AAV5 capsids. Therefore, applicants tested the localization of AAV2 and AAV5 capsids directly in the target tissue (i.e., the dog's knee joint) after intra-articular injection. Since HA expression is expected to be beneficial for both synovial cells and chondrocytes, vector genome copies were quantified in dog synovial and cartilage samples, respectively. The data showed that AAV2 provided highly inconsistent in vivo gene transfer to dog synovial and cartilage tissues and further showed little dose-response effect, for which the reasons are unclear. Similar experiments performed in OA rabbit joints demonstrated highly consistent rAAV2 vector genome detection using similar vector doses (Kyostio-Moore, 2015). In contrast to AAV2 vectors, AAV5 vector genomes were detected in a consistent manner in both tissue types (n=5 / group). Importantly, the detection of AAV5 in cartilage samples is surprising and unexpected, since cartilage has been reported to be difficult to transduce in vivo conditions due to extensive extracellular matrix, and thus far there have been no other reports of AAV5 detection in cartilage of many animals after intra-articular delivery.In addition, the dog study of the present disclosure produced unexpectedly high levels of rAAV5 vector in dog synovial tissue, which was also about 2 logs higher in synovium compared to that in cartilage, indicating a preference of AAV5 for the dog synovial lining.This preferential expression pattern could not have been predicted prior to the present disclosure as well. In addition to high levels of vector detection, recombinant HAS2 expression was confirmed in canine synovial and cartilage tissues by mRNA analysis, indicating that the CBA promoter is functional in both tissue types. Furthermore, detection of AAV5 transcripts in cartilage samples confirmed that chondrocytes were transduced by the vector and not by a virus hidden in the extracellular matrix of cartilage. For AAV2, similar levels of vector genome and transcripts were also observed in the synovial lining. However, AAV2 vector mRNA expression was surprisingly almost 100-fold lower than the corresponding vector genome detection in cartilage samples, suggesting that some of the vector persists outside of chondrocytes, possibly retained in the extracellular matrix. These significant differences will only be understood after the applicants perform significant non-routine experiments. Although the vector was administered to only one joint in each animal, vector genomes were occasionally detected in the contralateral, non-injected joints, mostly in synovial samples obtained from AAV2-treated joints, but none of the animals in which vector genomes were observed in the contralateral joints had any detectable HAS2 transcripts in these joints. In summary, the data disclosed herein demonstrate that AAV5 capsids provide successful gene transfer via intra-articular delivery to canine joints due to the low pre-existing humoral immunity to AAV5 in the subjects and the ability to transduce articular tissue following intra-articular injection, which can provide gene transfer not only to the synovial lining but also to the chondrocytes of the cartilage. Both Several tissue types will benefit from the increased HA synthesis afforded by the gene delivery compositions and methods of the present disclosure: the synovium will benefit from its enhanced ability to provide lubrication in synovial fluid, and cartilage will benefit by acting as a scaffold with enhanced matrix attachment and therefore improved cartilage health. These results suggest that overexpression of HA by AAV-mediated HAS2 gene transfer to diseased sites may alleviate OA pathology and pain. Decrease This indicates that. EXAMPLES

[0036] Construction and evaluation of lubricin AAV vectors Example 2a - Overview It has recently been shown that intra-articular injection of recombinant lubricin reduces cartilage degeneration in a rat OA model (Flannery, 2006). However, lubricin administered to the joint has a very short half-life in synovial fluid, with most of the protein being cleared within 72 hours (Vugmeyster, 2011). Thus, repeated intra-articular injections are required, which is time-consuming, laborious, stressful, and expensive. In contrast to HAS2 (see Example 1), lubricin is encoded by a large cDNA and contains multiple DNA repeats in its mucin-like domain, which make it difficult to fit into rAAV vectors and express at high levels, respectively. To circumvent this problem, applicants have generated truncated inulbricin cDNA and optimized a small expression cassette to enhance lubricin production. Importantly, neither the full-length inulbricin sequence nor the truncated forms disclosed herein were known prior to this disclosure. Briefly, Applicants generated a full-length inulbricin cDNA, which was then used to design a truncated, codon-optimized version of inulbricin (cLub1co). The latter was then used to construct various lubricin expression plasmids. These plasmids were characterized for lubricin mRNA and protein production after transfection of HEK293 cells. Data showed that each construct produced both lubricin mRNA and secreted lubricin. Finally, Applicants generated a rAAV vector carrying a cLub1 expression cassette, demonstrating the feasibility of rAAV / cLub1 vector production. HEK293 cells infected with this construct synthesized and secreted inulbricin.

[0037] Example 2b - Method Cloning of inulbricin: As there is no canine full-length lubricin cDNA in GenBank (incomplete sequence: GenBank no. ABD38836.1), the complete canine cDNA was obtained from a custom-synthesized canine cartilage cDNA library. To achieve this, overlapping fragments were generated using qPCR with diverse primers. The full-length cDNA (SEQ ID NO: 4) was then used to design a truncated form of inulbricin (cLub1), which resembles the shorter published version of human lubricin (Flannery, 2009). This shorter inulbricin contained a deletion of the sequence encoding amino acids 378 to 782. This truncated lubricin sequence was codon-optimized (cLub1co) and further synthesized (GeneArt / Invitrogen). The cLubco fragment (blunted KpnI to PmeI) was cloned into the Mfe (blunted)-PmeI site of a plasmid. The plasmid contained a CMV enhancer, a chicken β-actin promoter and truncated hybrid intron (Hib) (minCBA), and a bovine growth hormone (BGH) polyadenylation site. The ligation reaction was transformed into E. coli Stable II cells and grown at 30°C to minimize DNA rearrangements. The resulting clones were analyzed by restriction enzyme analysis and the cloning junctions were analyzed by DNA sequencing. Additional constructs were made that contained 6x histidine (6xHis) and modifications of two "ATG" sequences present in the intron sequence. The expression plasmids were used to analyze lubricin expression in vitro. Expression analysis of inulbricinHEK293 cells were transfected with the lubricin expression plasmid using Lipofectamine 2000 (Invitrogen) and cells were grown for 72 h. To analyze lubricin mRNA expression, cells were harvested and transcript levels were measured by real-time (RT) qPCR assays using BGA pA-specific primers / probes (7500 Real-Time PCR System; Applied Biosystems, Foster City, CA). For protein production analysis, culture fluids were harvested and concentrated approximately 20-30-fold (100k MWCO filter, Millipore). Samples were run on 4-12% Bis-Tris gels or 3-8% Tris-acetate (NuPAGE; Thermo Fisher Scientific) SDS-PAGE gels (reduced) in MOPS or Tris-acetate buffers, respectively. Lubricin was detected by Western blot using mouse anti-lubricin antibody (9G3, Millipore) (Ai, 2015) and goat anti-mouse-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) as the secondary antibody. Preparation of AAV / cLub1co The cLub1 expression cassette was cloned into an AAV inverted terminal repeat (ITR)-containing plasmid to generate an expression cassette flanked by AAV ITRs (previral plasmid pDC627), constructing psITR / minCBA-HI-cLub1co-BGHpA. To test the packaging of the cLub1 expression cassette-containing plasmid, 293 cells were cultured at 8x10 5Cells / well were seeded (6-well plates) and transfected the following day with psITR / minCBA-HI-cLub1co-BGHpA, or psp70 / EGFP, pHLP-19cap2 (AAV2) or p5repCMVcap5 (AAV5) plasmids and pAdHELP (Promega CaPO4kit) to package the vector into AAV2 or AAV5 capsids. Cells were harvested 3 days later and lysates were quantified for vector yield by qPCR assay (7500 Real-Time PCR System). BGH pA sequence-specific primers / probe (Applied Biosystems / Life Technologies) and a standard curve of serially diluted linearized plasmid DNA (containing BGH pA) were used. rAAV virus yield was expressed as the amount of DNase-resistant particles (DRP) per cell (Clark, 1999). Laboratory-scale vector production was performed using triple transfection of psITR / minCBA-HI-cLub1co-BGH, pHLP19-cap5 (for AAV5 vectors), and pAdHELP. Vectors were purified by CsCl gradient and yields quantified as described above (University of Massachusetts Medical School, Worcester, MA).

[0038] Example 2c - Results Creation of short inulbricin : Because the inulbricin sequence present in GenBank is missing a large portion of exon 6 (encoding 857 amino acids), a full-length inulbricin cDNA (4017 bp (not including the stop codon), SEQ ID NO: 4) was generated. The full-length inulbricin cDNA encodes a protein (SEQ ID NO: 5, FIG. 9) containing a total of 1339 amino acids (slightly smaller than the human sequence of 1404 amino acids). At the amino acid level, this inulbricin shares 79% identity with the sequence of human lubricin (SEQ ID NO: 11, FIG. 16). Because full-length inulbricin is too large to fit into a rAAV vector due to packaging limitations, a truncated version of inulbricin was generated. This short inulbricin version ("Lub1") was generated by deleting the sequence encoding amino acids 378 to 782 of the mucin-like domain, resulting in a 2949 bp long cDNA (SEQ ID NO:6) encoding 983 amino acids (SEQ ID NO:7). Despite the large deletion of the mucin-like domain, approximately 10 KEPAPTT-like peptide repeats remained. Importantly, none of these are identical to the canonical human repeat sequences, but even if they were identical, a person skilled in the art would not have predicted that delivery of this truncated canine Lub1 would be effective in treating OA. These repeats are thought to be important for lubricating properties as they are potential O-linked oligosaccharide binding sites. Codon optimization of this short lubricin (Lub1co, SEQ ID NO:6) increased the GC content from 44% to 60% and had 74% nucleotide similarity with the native canine DNA sequence. This short canine cDNA was then used to generate a plasmid expression cassette with the minCBA promoter, cLub1co and BGHpA (FIG. 10). An expression plasmid with a 6xHis-tag and modification of the putative ATG nucleotide sequence within the intron region (to minimize false translation start sites) was also generated. Expression analysis of inulbricinExpression of cLub1co from the minimal CBA promoter (minCBA-cLub1co) plasmid was confirmed in vitro by showing increased mRNA levels in transfected 293 cells (Figure 11A). Activity of the minCBA-cLub1co construct with a short intron was approximately 3-fold lower than with the full-length CBA-HI construct (CBA-cLub1co). Almost no transcription was observed with the plasmid containing full-length lubricin and the non-codon-optimized construct (CBH-cLubr). Transcript analysis was also performed for expression cassettes with various modifications (Figures 10, 11B). Expression of minCBA-Lub1co was comparable to the EGFP and C-terminal 6xHis-tag containing constructs. Deletion of the two putative ATG codons present in the hybrid intron appeared to enhance expression levels approximately 2-fold. Other morphological changes observed in Lubico transfected cells also suggested Lub1 expression. This is because these changes were not present in non-transfected or EGFP plasmid-transfected cells (data not shown). Production of canine Lub1 protein from various expression plasmids was examined by Western blot with an antibody against lubricin, showing a protein of 250-380 kDa in concentrated culture fluids (Figure 12A). The expected size based on 1339 amino acids is approximately 160 kDa, but the larger and more spread-out pattern signal is likely due to glycosylation. Little detection was observed in non-transfected or EGFP plasmid-transfected cells. In addition, the ΔATG modification appeared to increase lubricin detection, similar to the observation for the increased transcript levels derived from this construct. Protein expression was also confirmed from the previral AAV plasmid, showing comparable protein detection (Figure 12B). In summary, these results demonstrated that the plasmid containing the inulbricin expression cassette expresses and secretes glycosylated lubricin protein. Generation of rAAV vector containing an inulbricin expression cassetteHaving confirmed inulbricin expression in the plasmid vector, we next tested in small-scale packaging experiments whether the expression cassette could be packaged into AAV2 and AAV5 capsid serotypes (Figure 13A,B). The data showed similar packaging efficiency of inulbricin in both AAV2 and AAV5 capsids as observed with the EGFP-expressing vector. The addition of the 6xHis-tag did not alter rAAV vector yields. Approximately 5-fold lower levels of packaging were measured with the AAV2 vector containing the canine HAS2 expression cassette. Laboratory-scale production of AAV5 / minCBA-cLub1 was then performed to evaluate scale-up vector production. Vector yields were comparable to standard AAV2 and AAV5 vector yields containing EGFP as a transgene (data not shown). AAV5 vectors were then tested in vitro for lubricin production and secretion in HEK293 cells. Analysis of conditioned media by Western blot showed dose-dependent detection of inulbricin (Figure 14). In summary, the data demonstrated that a truncated version of inulbricin can be used to generate rAAV vectors, and that cells infected with this vector can mediate lubricin synthesis and secretion into the medium.

[0039] Example 2d - Conclusion As shown above, lubricin as a transgene presents many problems for rAAV production. First, the size of lubricin cDNA containing the necessary expression elements exceeded the packaging capacity of rAAV, and therefore a shorter cDNA was required. Interestingly, compared to the human lubricin amino acid sequence of the mucin-like domain, the complete KEPAPTT repeat is absent in the canine sequence (Figure 16). For the production of recombinant rAAV vectors, any repeat of DNA sequence may pose a challenge, since repeat sequences may reduce the stability and integrity of the viral genome by inducing DNA deletions and rearrangements during virus production. However, given that comparable vector yields were obtained when compared to standard EGFP reporter vectors, the disclosed (and surprising) results showed that the production of rAAV viruses containing and expressing the novel inulbricin sequence is feasible. Thus, this is the first report to reveal a single rAAV vector strategy for lubricin gene delivery. EXAMPLES

[0040] In vivo efficacy study of AAV-HAS2 in a medial meniscal ligament release (MMR) model The objective of this study was to evaluate the efficacy of HA synthase 2 gene therapy in a canine OA stifle model using gross observations and histology.Twelve intact laboratory breed male mixed breed dogs (foxhound phenotype, approximately 20-23 kg) were anesthetized and underwent arthroscopic medial meniscal ligament release (MMR) of the right stifle joint (day -14). Phosphate-buffered saline (PBS control) or recombinant AAV5 DNase-resistant particles (DRP) (5x10 11 ) was administered intra-articularly on day 0 (n=6 / group). For biomarkers of joint inflammation, plasma was collected from all dogs on days 0 and 182. For analysis of HA levels, right and left synovial fluid was collected on days 0, 56, 112 and 182 from all PBS control and cHAS-2 treated groups. On day 182, dogs were euthanized, stifle ligament release-induced cartilage defects (visible by India ink staining) were measured, and joint tissue was collected for histopathological examination according to OA Research Society International (OARSI) standard techniques. Macroscopic and histological data were analyzed by Kruskal-Wallis analysis using GraphPad Prism 6 statistical software. Measurement of total HA levels in synovial fluid did not reveal any treatment-related differences in total HA levels (Figure 17). Synovial and cartilage samples were collected from treated joints on day 182 and analyzed for detection of viral genomes (Figure 18A). Vector-derived DNA and mRNA were detected in synovial samples (Figure 18A) and most cartilage samples (Figure 18B) from each individual rAAV5 / cHAS-2 injected joint. The data are summarized in Figure 18C, showing the average vector genome and mRNA in both tissue samples. There was no evidence of local or systemic toxicity associated with intra-articular administration of HA synthase-2 gene therapy. There was consistent preservation of cartilage architecture with cHAS-2 treatment compared with PBS treatment. Reductions in size and depth of lesions on both the medial femoral condyle and medial tibial plateau articular surfaces were more evident in the femoral condyles in four of six rAAV5 / cHAS2-treated dogs. In FIG. 19, the histopathology scores according to Cook et al. (Cook et al., 2001) are shown in the lower left corner of each of the medial femoral condyle and medial tibial cartilage images (2x). Dog 994731 / PBS had extensive erosion extending towards the medial zone with significant proteoglycan loss at both cartilage surfaces. No chondroprotective effect was observed. Dog 993107 (rAAV5 / cHAS2 treated) had shallow lesions in the superficial zone of the femoral cartilage, but overall there was good preservation of the remaining cartilage with little loss of proteoglycan. The tibial plateau lesions were deeper towards the medial zone with mild loss of proteoglycan. As the underlying cartilage was relatively normal, chondroprotective effect was present at the femoral condyle. Dog 992879 treated with rAAV5 / cHAS2 showed some proteoglycan loss in the femoral cartilage, but the overall morphology was preserved. The tibial plateau had well-defined focal erosions, but the majority of the cartilage was preserved, thus demonstrating some degree of chondroprotection, as the lesions were small and of minor severity. Notably, one of the rAAV5-treated animals in which the vector was undetectable in cartilage samples also had the largest tibial plateau lesion area (dog 993107, FIG. 19). Conversely, synovial and cartilage lesions both One of the rAAV5-treated animals (dog 992879), in which vector was detected in the cartilage (but no mRNA was detected in the cartilage), had the best cartilage structure. Thus, the presence of the rAAV5-HAS2 vector correlated with the best cartilage structure, whereas absence of the vector correlated with the greatest tibial plateau lesion area. Thus, despite variability in vector / mRNA detection, the rAAV5 vector expressing HAS2 appears to have induced the desired clinical outcome. Taken together, these results demonstrated consistent rAA5-mediated gene transfer to the synovium and cartilage of canine OA joints and confirmed sustained vector-derived expression for at least 6 months. Histological analysis showed a reduction in cartilage lesions and slowed disease progression in the majority of cHAS-2-treated joints, while little difference was observed in total synovial HA levels. The latter may indicate that localized HA expression in cartilage and synovium may have some disease-modifying properties without increasing total synovial HA levels. Alternatively, molecular weight changes in synthesized HA, undetectable by total HA level measurements, may also have contributed to the beneficial effects of rAAV5 / cHAS-2.

[0041] References Sanderson RO et al.Systematic review of the management of canine osteoarthritis.Veterinary Record (2009) 164, 418-424 McIlwraith CW.Frank Milne Lecture: from arthroscopy to gene therapy: 30 years of looking in joints.Am Assoc Equine Pract 2005;51:65-113. Cook et al.The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the dog.Osteoarthritis Cartilage, 2010;18 suppl 3:S66-79.

[0042] The invention is further represented by the following numbered paragraphs: 1. A method for treating a subject mammal suffering from osteoarthritis (OA), comprising the step of administering to the subject mammal a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) intra-articularly, the recombinant adeno-associated virus comprising a nucleic acid encoding an osteoprotective or bone regenerative polypeptide operably linked to a promoter, the polypeptide being expressed in vivo in the subject mammal in an amount effective to alleviate a symptom of OA. 2. The method of claim 1, wherein the polypeptide is hyaluronic acid synthase (HAS), lubricin, interleukin-1 receptor (IL-1R) antagonist, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2), transforming growth factor beta 1 (TGFβ1), bone morphogenetic protein 7 (BMP7), glucosamine-fructose-6-phosphate aminotransferase (GFAT), interleukin 10 (IL-10), heme oxygenase-1 (HO-1), biologically active truncated forms thereof, or combinations thereof. 3. The method according to item 1 or 2, wherein the polypeptide is a HAS2 polypeptide. 4. The method according to any one of items 1 to 3, wherein the target mammal is a human, a dog, or a cat. 5. The method according to any one of items 1 to 4, wherein the target mammal is a dog. 6. The method according to item 5, wherein the polypeptide is canine HAS2. 7. The method according to item 5 or 6, wherein the HAS2 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:2, or a fragment, variant or homologue thereof which exhibits HAS2 in vivo activity in the subject animal. 8. The method according to any one of items 5 to 7, wherein the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO:2. 9. The method according to any one of items 5 to 8, wherein the nucleic acid encoding the HAS2 polypeptide has a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:3. 10. The method of any of items 5-9, wherein the rAAV comprises an rAAV vector genome comprising the following elements from 5' to 3': a 5' AAV inverted terminal repeat (ITR), a stuffer nucleic acid, a promoter, an intron (IN), a cHAS2 codon-optimized cDNA, a polyadenylation signal (pA), and a 3' AAV ITR. 11. The method according to item 10, wherein the promoter is a chicken beta-actin (CBA) promoter. 12. The method of claim 1 or 2, wherein the polypeptide is a lubricin polypeptide. 13. The method of item 12, wherein the lubricin polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:7, or a fragment, variant, or homologue thereof that exhibits lubricin activity in vivo in the subject animal. 14. The method of claim 13, wherein the lubricin polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7. 15. The method of claim 13 or 14, wherein the nucleic acid encoding the lubricin polypeptide has a nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in SEQ ID NO:6. 16. The method of any of items 13-14, wherein the rAAV comprises a rAAV vector genome encoded by the plasmid pITR / minCBA-HI-cLub1co-BGH. 17. The method according to any of items 1-9 or 12-16, wherein the promoter is selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, an adenovirus major late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a beta-actin gene promoter, a CBA promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase (CK) gene promoter. 18. The method of item 1, wherein the AAV comprises an AAV2 or AAV5 capsid. 19. A method for increasing the production of hyaluronic acid in canine chondrocytes and / or synovial cells, comprising the step of administering to the dog an rAAV, wherein the rAAV comprises an rAAV vector genome comprising a nucleic acid encoding an HAS2 enzyme operably linked to a promoter, and following administration, the HAS2 enzyme is expressed and catalyzes the production of additional hyaluronic acid, thereby increasing the levels of hyaluronic acid (HA) in the dog. 20. The method of item 19, wherein HAS2 is produced in an amount sufficient to treat symptoms of OA in a dog. 21. The method according to item 20, wherein HA levels are restored to levels found in healthy dogs. 22. A method for treating a dog suffering from OA, comprising administering to the dog a therapeutically effective amount of rAAV, wherein the rAAV comprises an AAV vector genome comprising a nucleic acid encoding HAS2 operably linked to a promoter. 23. A method for treating a human suffering from OA, comprising administering to the human a therapeutically effective amount of rAAV, wherein the rAAV comprises an AAV vector comprising a nucleic acid encoding HAS2 operably linked to a promoter. 24. The method according to any one of items 19-23, wherein the nucleic acid encoding HAS2 has at least 90% identity to the nucleotide sequence shown in SEQ ID NO:3 or encodes a HAS2 having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2. 25. The method according to any one of items 19 to 23, wherein HAS2 has the amino acid sequence shown in SEQ ID NO:2. 26. A method for increasing production of lubricin in canine chondrocytes and / or synovial cells, comprising the step of administering to the dog an rAAV, wherein the rAAV comprises an rAAV vector genome comprising a nucleic acid encoding lubricin operably linked to a promoter, such that following administration, the lubricin is expressed, thereby increasing the levels of lubricin in the dog. 27. The method of item 26, wherein lubricin is produced in an amount sufficient to treat symptoms of OA in a dog. 28. The method of item 26, wherein lubricin levels are restored to levels found in healthy dogs. 29. A method for treating a dog suffering from OA, comprising administering to the dog a therapeutically effective amount of rAAV, wherein the rAAV comprises a rAAV vector genome comprising a nucleic acid encoding lubricin operably linked to a promoter. 30. A method for treating a human suffering from OA, comprising administering to the human a therapeutically effective amount of a rAAV, wherein the rAAV comprises an AAV vector genome comprising a nucleic acid encoding lubricin operably linked to a promoter. 31. The method of any of items 26-30, wherein the nucleic acid encoding the lubricin polypeptide has at least 90% identity to the sequence set forth in SEQ ID NO:6, or the nucleic acid encodes a lubricin having an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7. 32. The method according to any one of items 26-30, wherein lubricin has the amino acid sequence shown in SEQ ID NO: 7. 33. The method according to any of items 19-32, wherein the promoter is selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter. 34. The method of any of items 19-25, wherein the rAAV comprises a rAAV vector genome encoded by the plasmid Ps-AAV-ITR / CBA-cHAS2co-BGH. 35. The method of any of items 26-32, wherein the rAAV comprises a rAAV vector genome encoded by the plasmid Ps-AAV-ITR / minCBA-HI-cLub1co-BGH. 36. A method for preventing the onset of OA in a subject mammal at risk thereof, comprising administering to a dog a therapeutically effective amount of rAAV, wherein the rAAV comprises an rAAV vector genome comprising a nucleic acid encoding HAS2 operably linked to a promoter. 37. The method according to item 36, wherein the nucleic acid encoding the HAS2 polypeptide has at least 90% identity to the sequence shown in SEQ ID NO:2 or encodes a HAS2 having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2. 38. The method according to item 36 or 37, wherein the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO:3. 39. A method for preventing the onset of OA in a subject mammal at risk thereof, comprising administering to a dog a therapeutically effective amount of a rAAV, wherein the rAAV comprises an rAAV vector genome comprising a nucleic acid encoding lubricin operably linked to a promoter. 40. The method of item 39, wherein the nucleic acid encoding lubricin has at least 90% identity to the sequence set forth in SEQ ID NO:6, or the nucleic acid encodes a lubricin having an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7. 41. The method of item 40, wherein the lubricin polypeptide has the amino acid sequence shown in SEQ ID NO: 7. 42. The method according to any of items 36-38, wherein the promoter is selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter. 43. The method of item 26, wherein the rAAV comprises a rAAV vector genome encoded by the plasmid Ps-AAV-ITR / CBA-cHAS2co-BGH. 44. The method of item 26, wherein the rAAV comprises a rAAV vector genome encoded by the plasmid Ps-AAV-ITR / minCBA-HI-cLub1co-BGH. 45. The method according to any of items 19-44, wherein the rAAV is administered intra-articularly. 46. ​​A recombinant plasmid vector comprising a nucleic acid sequence encoding a canine HAS2 polypeptide operably linked to a promoter. 47. The recombinant plasmid according to item 46, wherein the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity to the sequence set forth in SEQ ID NO:3, or the nucleic acid encodes a HAS2 polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2. 48. The recombinant plasmid according to item 46 or 47, wherein the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. 49. A recombinant plasmid according to any one of items 46-49, comprising pCBA-HI-cHAS2-BGHpA. 50. A recombinant plasmid vector comprising a nucleic acid sequence encoding a truncated inulbricin operably linked to a promoter. 51. The recombinant plasmid of item 50, wherein the nucleic acid sequence encoding lubricin has at least 90% identity to the nucleotide sequence set forth in SEQ ID NO:6, or the nucleic acid encodes a lubricin comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7. 52. The recombinant plasmid according to item 50 or 51, wherein the lubricin polypeptide has the amino acid sequence shown in SEQ ID NO: 7. 53. The recombinant plasmid according to any of items 46-49 or 50-52, wherein the promoter is selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter. 54. A recombinant AAV5 viral vector comprising the nucleotide sequence shown in SEQ ID NO:8. 55. An rAAV comprising the rAAV vector described in item 53. 56. A pharmaceutical composition comprising the rAAV according to item 55 and at least one pharma- ceutical or veterinarily acceptable carrier, excipient or vehicle. 57. A method for treating a subject mammal suffering from osteoarthritis, comprising the step of intra-articularly administering to the subject mammal a therapeutically effective amount of the pharmaceutical composition described in item 56. 58. The method according to item 57, wherein the target mammal is a human or a dog. 59. An adeno-associated virus (AAV)-based biological delivery and expression system for use in the treatment of OA of mammalian joints by long-term gene expression of HAS2 or lubricin in synoviocytes and / or chondrocytes, said system comprising a rAAV, said rAAV comprising an rAAV vector comprising a nucleic acid sequence encoding HAS2 or lubricin, left and right AAV inverted terminal repeats (L ITR and R ITR), wherein expression of the HAS2 or lubricin gene in the synoviocytes and / or chondrocytes is regulated by a promoter, said promoter being positioned upstream of the reading frame of the nucleic acid sequence encoding HAS2 or lubricin, and further wherein said promoter is specifically activated by increased levels of an immune stimulant. 60. The AAV system according to item 59, wherein the HAS2 is a mammalian HAS2. 61. The AAV system according to item 59 or 60, wherein HAS2 is human HAS2. 62. The AAV system according to any of items 59-61, wherein the promoter is an inflammation-inducible promoter. 63. The AAV system according to item 62, wherein the inflammation-inducible promoter is selected from the group consisting of NF-KB promoter, interleukin 6 (IL-6) promoter, interleukin-1 (IL-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1a (MIP-1a) promoter and hybrid constructs thereof. 64. The AAV system according to any of items 59-63, wherein the rAAV vector genome comprises a nucleic acid encoding HAS2 comprising the amino acid sequence of SEQ ID NO:2, lubricin comprising the amino acid sequence of SEQ ID NO:7, or a functional variant thereof. 65. The AAV system according to any of items 59-64, wherein the rAAV vector genome comprises a marker gene which allows monitoring of the vector genome in synovial cells and / or chondrocytes. 66. The AAV system according to any of items 59-65, wherein the rAAV vector genome comprises a nucleic acid having at least 80% or 90% sequence identity to the nucleic acid sequence shown in SEQ ID NO:3 or SEQ ID NO:6. 67. The AAV system according to any of items 59-66, wherein the rAAV vector genome comprises the nucleic acid sequence shown in SEQ ID NO:3 or SEQ ID NO:6. 68. The AAV system according to any of items 59-67 for the treatment or prevention of osteoarthritis. 69. A pharmaceutical composition comprising an AAV system according to any of items 59-68. 70. An rAAV comprising an rAAV vector, the rAAV comprising a nucleic acid sequence encoding a canine HAS2 polypeptide operably linked to a promoter. 71. The rAAV according to item 70, wherein the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity to the sequence set forth in SEQ ID NO:3, or the nucleic acid encodes a HAS2 polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2. 72. The rAAV according to item 70 or 71, wherein the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. 73. An rAAV comprising an rAAV vector comprising a nucleic acid sequence encoding inulbricin operably linked to a promoter. 74. The rAAV of item 73, wherein the nucleic acid sequence encoding lubricin has at least 90% identity to the nucleotide sequence set forth in SEQ ID NO:6, or the nucleic acid encodes lubricin comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7. 75. The rAAV according to item 73 or 74, wherein the lubricin polypeptide has the amino acid sequence shown in SEQ ID NO: 7. 76. The AAV according to any one of items 50-53, wherein the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO:8. 77. The rAAV according to any of items 71-72 or 74-76, wherein the promoter is selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter. 78. The rAAV according to any of items 71-77, comprising an AAV2 capsid or an AAV5 capsid. 79. A pharmaceutical composition comprising an rAAV according to any of items 71-78 and at least one pharma- ceutical or veterinarily acceptable carrier, excipient or vehicle. 80. A method for treating a subject mammal suffering from osteoarthritis, comprising the step of intra-articularly administering to the subject mammal a therapeutically effective amount of the pharmaceutical composition described in item 79. 81. The method according to item 80, wherein the target mammal is a human or a dog. 82. An isolated nucleic acid having the sequence shown in SEQ ID NO:4. 83. An isolated polypeptide having the sequence shown in SEQ ID NO:5. The invention will now be defined according to the following non-limiting claims.

Claims

1. 1. A pharmaceutical composition for use in a method of treating a mammalian subject suffering from osteoarthritis comprising administering into a joint of the mammalian subject a recombinant adeno-associated virus (rAAV) vector comprising a single-stranded DNA genome comprising a nucleic acid sequence encoding a truncated inulbricin polypeptide operably linked to a promoter, wherein the rAAV comprises an rAAV vector comprising a single-stranded DNA genome comprising a nucleic acid sequence encoding a truncated inulbricin polypeptide operably linked to a promoter, wherein the truncated inulbricin polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence comprising at least one O-linked glycosylation site, the rAAV comprises an AAV5 capsid, and the polypeptide is expressed in vivo in an amount effective to alleviate a symptom of osteoarthritis in the subject mammal.

2. 2. The pharmaceutical composition of claim 1, wherein the truncated inulbricin polypeptide has the amino acid sequence shown in SEQ ID NO:

7.

3. The pharmaceutical composition of claim 1 or 2, wherein the nucleic acid sequence encoding the truncated inulbricin polypeptide has a nucleic acid sequence having at least 90% identity to the nucleic acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence encoded by the nucleic acid sequence contains at least one O-linked glycosylation site.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein at least one O-linked glycosylation site comprises a KEPAPTT-like repeat.

5. The pharmaceutical composition described in any one of claims 1 to 4, wherein the promoter is selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter.

6. The pharmaceutical composition of any one of claims 1 to 4, wherein the single-stranded DNA genome comprises the following elements from 5' to 3': a 5' AAV inverted terminal repeat (ITR), a promoter, an intron (IN), a truncated inulbricin codon-optimized cDNA, a polyadenylation signal (pA) and a 3' AAV ITR.

7. A recombinant adeno-associated virus (rAAV) vector comprising a single-stranded DNA genome comprising a nucleic acid sequence encoding a truncated inulbricin polypeptide operably linked to a promoter, said truncated inulbricin polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:7, said amino acid sequence comprising at least one O-linked glycosylation site, and comprising an AAV5 capsid.

8. The rAAV of claim 7, wherein the nucleic acid sequence encoding the truncated inulbricin polypeptide has a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO:

6.

9. The rAAV of claim 7 or 8, wherein the truncated inulbricin polypeptide comprises the amino acid sequence shown in SEQ ID NO:

7.

10. The rAAV of any one of claims 7 to 9, wherein the promoter is selected from the group consisting of a CMV IE promoter, an RSV promoter, an HSV-1 TK promoter, an SV40 early promoter, an SV40 late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an alpha-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a beta-actin gene promoter, a beta-globin gene promoter, a gamma-globin gene promoter, an alpha-fetoprotein gene promoter, and a muscle creatine kinase gene promoter.

11. The rAAV of any one of claims 7 to 9, wherein the single-stranded DNA genome comprises an expression cassette comprising the following elements from 5' to 3': a chicken β-actin promoter, a hybrid intron, a nucleic acid sequence encoding a truncated inulbricin polypeptide, and a bovine growth hormone polyA site.

12. A pharmaceutical composition comprising an rAAV described in any one of claims 7 to 11.

13. 13. The pharmaceutical composition of claim 12, further comprising at least one pharma- ceutically or veterinarily acceptable carrier, excipient or vehicle.

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