Recombinant AAV vector expressing bone-protective genes including HAS2 and rubrisin, useful for the treatment of mammalian osteoarthritis and related joint symptoms.

Abstract

To provide recombinant viral vectors, pharmaceutical compositions comprising such recombinant vectors, and methods for prevention and treatment of osteoarthritis in mammals.SOLUTION: This disclosure provides adeno-associated virus (AAV) vectors capable of expressing, in a host, osteoprotective / chondroprotective bioactive proteins, including hyaluronan synthase 2 (HAS2) and lubricin (PRG4). Methods of production of these AAVs are provided, as are methods of treatment of osteoarthritis in mammalian joints, by the long-term gene expression of osteoprotective / chondroprotective proteins, including HAS2 and PRG4, in both synovial and chondrocyte cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority over U.S. Provisional Patent Application No. 62 / 278,243 (filed January 13, 2016 (the entire application of which is incorporated herein by reference)). (Description of Sequence Listing) The sequence listing accompanying this application is provided in text format instead of as a printout copy, and is incorporated herein by reference. The name of the text file containing the sequence listing is MER 16-291 SEQ Listing_ST25.txt. The text file is 57.6KB and was created on January 13, 2016. It was filed electronically via EFS-Web at the same time as 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 bioactive polypeptides belonging to the hyaluronansynthase 2 (HAS2) and rubrisin (PRG4) family proteins in a host. Accordingly, the present invention relates to the field of genetic engineering and provides adeno-associated virus (AAV)-based biological delivery and expression systems used for 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 the joints of mammals 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 strong connective tissue that covers the ends of bones in the joints. It provides a highly lubricated surface with relatively little friction between the hard bones, allowing for smooth movement. As OA progresses, the cartilage is partially or completely lost due to abnormal or excessive wear, leading to exposure of the bone ends, which then rub against each other, resulting in inflammation, pain, swelling, or decreased mobility. Currently, the detailed reasons for the initial chondropenia leading 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 gait, with an estimated 20 percent of dogs over one year of age affected. Currently, there is no available treatment that provides a cure for OA, and therefore medical treatment is largely focused on symptom relief rather than cartilage regeneration. Pain relief treatment typically involves steroids and non-steroidal anti-inflammatory drugs (NSAIDS), which have shown effectiveness in treating OA for decades. However, while these drugs can suppress inflammation in the joints, many are known to have degenerative effects on cartilage (this effect further exacerbates the underlying process of OA progression). In addition to traditional analgesic and anti-inflammatory therapies, direct administration of naturally occurring bone-protective compounds has been used to alleviate OA symptoms, but with varying degrees of success. For example, hyaluronic acid (HA) has been widely used to restore viscoelasticity and lubrication in affected joints. Furthermore, polysulfated glycosaminoglycans (PSGAGs), administered via intra-articular or intramuscular routes, as well as orally administered glucosamine and chondroitin sulfate, have shown some efficacy.

[0003] However, the aforementioned drugs must be administered frequently (sometimes in combination) to achieve meaningful symptom relief. These frequent joint injections are labor-intensive / expensive, carry a risk of infection, and cause considerable stress to the patient or animal. Surgical approaches have also been developed, but they generally show low efficacy in dogs and horses and are typically performed only in severely ill animals in the advanced stages of the disease. In addition to the delivery of supplements / medicines, several groups have attempted to improve OA symptoms by delivering polypeptides or nucleic acids that can express in the host means of producing viscoelastic / viscoprotective polypeptides, nucleic acids encoding them, or viscoprotective proteins (e.g., enzymes). Approaches of greater interest include the use of lubricin polypeptide (Flannery, US 7,642,236 B2), tribonectin (US 7,618,914 B2 (Rhode Island General Hospital)), and hyaluronansynthase (US 6,423,514 (Millennium Pharmaceuticals)). Some of these attempts can be characterized as “gene therapy” (the basic concept is 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, a group 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 expressed human interferon beta using AAV5 in the context of rheumatoid arthritis (RA) (to reduce inflammatory cytokines). Unlike OA, inflammatory signaling plays a crucial role in the pathology of RA, and therefore, blocking this signal is an important therapeutic approach. However, another group pursuing possible links between inflammation and OA expressed an IL1 receptor antagonist using recombinant AAV2 in the case of equine OA (Goodrich et al., Molecular Therapy-Nucleic Acids (2013) 2, e70). [Overview of the project] [Problems that the invention aims to solve]

[0005] However, none of these approaches have been proven universally effective, and there remains a serious unmet need regarding the alleviation of pain and suffering in OA patients. As a result, there is a clear and unmet medical need for a more effective, sustainable, and cost-effective treatment in the long run. Accordingly, as described in detail herein, we are the first to demonstrate that recombinant adeno-associated virus (rAAV) vectors can deliver cDNA encoding a therapeutic substance by a single intra-articular injection into a mammalian joint, thereby promoting local and continuous in vivo production of the substance in synovial and chondrocytes. The applicants also isolated and sequenced the full-length inulbricin cDNA (SEQ ID NO: 4) for the first time. [Means for solving the problem]

[0006] The present invention provides an rAAV vector that expresses therapeutically effective amounts of bone protection genes and / or bone regeneration gene products in vivo in a mammalian host. In several features, the rAAV may include cDNA encoding a substance having disease-modifying properties, lubrication properties, anti-inflammatory properties, and pain-relieving properties.

[0007] In several features, the rAAV vector is derived from AAV serotypes 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 contains an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV2rAAV6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In yet another embodiment, the rAAV particle contains a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In some embodiments, the ITR and capsid are derived from the same AAV serotype. In other embodiments, the ITR and capsid are derived from different AAV serotypes. In some embodiments, the rAAV vector may be an AAV2 or AAV5 capsid serotype. In some related embodiments, the rAAV2 and rAAV5 vectors include at least one ITR derived from AAV2. In multiple feature embodiments, the rAAV vector encodes hyaluronic acid 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. Glycosaminoglycan hyaluronanate (HA) is a non-sulfated glycosaminoglycan consisting of repeating glucuronic acid and N-acetylglucosamine residues linked by beta-1-3 and beta-1-4 glycosidic bonds. HA provides 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 is found in tissues such as connective tissue, epithelial tissue, and nerve tissue. In the joint space, HA is produced by chondrocytes along with 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. Lower molecular weight HA (<500kDa) is closely related to receptor-mediated activation of angiogenesis, malignancy, and inflammation, while higher molecular weight HA provides lubrication in joints.

[0008] The size and concentration of articular HA are both regulated by their synthesis and degradation rates. HA degradation is mediated by hyaluronidases, which cleave HA into smaller fragments, which are then excreted into the lymphatic system for removal. Three enzymes (referred to as hyaluronan synthases (HAS) 1, 2, and 3) have been described for HA production, and these are located on the inner surface of the synovial cell plasma membrane. Of these, HAS2 has been shown to be necessary for the production of high molecular weight HA (Itano et al., 1999). High molecular weight HA levels are reduced in osteoarthritis in both human patient and animal OA models (Plickert et al., 2013). This is likely due to decreased HA synthesis and increased HA degradation by hyaluronidases. Of the HA synthases, HAS2 and 3 are expressed in human cartilage, and of these, HAS2 expression is reduced in human OA. HA levels are also reduced 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 pre- and anti-inflammatory mediators, with conflicting effects reported. These mediators include TGFβ, primary epidermal growth factor, TNF-alpha, and retinoic acid (Guo, Kanter el al., 2007; Hyc et al., 2009). Downregulation of diseased joints by inflammatory mediators is expected to reduce HAS2 expression, leading to decreased HA levels and potentially discriminatively affecting various HAS isoforms (David-Raoudi et al., 2009). In contrast, mechanical stimulation (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 both pericellular presence and secretion into the extracellular space. It is unclear what regulates the degree of secretion. However, typically about 80% of HA is secreted, while the remainder remains bound to the producing cells.This cell-bound HA is crucial for the assembly of matrix proteins, and blocking HAS2 synthesis leads to a decrease in the cell-bound matrix and an increase in the release of proteoglycans (i.e., agrecans) into the medium, further confirming the major role of HAS2 as the key enzyme for HA synthesis in chondrocytes (Nishida et al., 1999).

[0009] In some embodiments of multiple features, the rAAV vector encodes rubrisin or a variant thereof. In some embodiments, rubrisin is human rubrisin. In other embodiments, rubrisin is inulubrisin. In some embodiments, rubrisin is codon-optimized rubrisin. Lubricin (PRG4) is a large mucin glycoprotein produced by synovial lining cells and chondrocytes of cartilage, providing lubrication that protects the cartilage surface (Flannery 1999; Schmidt 2001; Waller 2013). Along with HA, lubricin is also an important lubricant in synovial fluid and provides shock absorption properties. Lubricin deficiency in mouse models and in rare genetic disorders in humans leads to cartilage degeneration characteristic of osteoarthritis (OA) (Rhee 2005; Ruan 2013). Reduced lubricin synthesis 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 an AAV2 or AAV5 capsid serotype encoding canine codon-optimized hyaluronic acid synthase-2 (HAS2). In several embodiments, the rAAV vector is administered by intra-articular delivery. In several embodiments, rAAV is administered by a single intra-articular delivery. In another embodiment, following intra-articular administration, in vivo production and secretion of the homologous therapeutic substance derived from the rAAV transduced cells can persist for at least about 6 months. In some embodiments, the rAAV vector includes an expression cassette containing a ubiquitous promoter and a codon-optimized species-compatible transgene (see Figure 1A). In another feature, this disclosure provides a method for expressing bone protection and / or bone regeneration gene products in vivo in animal joints using an rAAV vector. All references cited herein (including patent applications and patent publications) are incorporated herein by reference in their entirety. The following detailed description (provided as an example, but not intended to limit the invention to the specific embodiments described) will be best understood in conjunction with the accompanying drawings. The accompanying drawings are as follows: [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A is a schematic diagram showing the HAS expression cassette in a plasmid (top panel) and an rAAV virus vector (bottom panel). [Figure 1B] Figure 1B is a graph showing HA production in cells transfected with the cHAS2 expression plasmid. 293 cells were transfected with the cHAS expression plasmid, and conditioned culture media were collected 3 days later. HA levels in the culture medium were quantified using a detection system with HA-binding proteins. Abbreviations: CBAcHAS2, HAS2 expression plasmid; EGFP, EGFP expression plasmid; untransfected, negative control cells; "Optimem" = cells grown in serum-free medium; "Complete" = cells grown in serum-containing medium. [Figure 1C] Figure 1C is an agarose gel image used for component separation of conditioned culture medium (24-hour hyaluronidase treatment + or -) of cHAS-transfected 293 cells, where HA expression is confirmed. Abbreviations: CM, conditioned culture medium; MDa, molecular weight size of the marker. [Figure 2A]Figure 2A is a graph showing rAAV vector production by small-scale packaging of cHAS2 into rAAV2 and AAV5 vectors using the triple transfection method. Packaging of the EGFP expression cassette into AAV2 is shown as a positive control, and packaging of cHAS in the absence of the capsid plasmid is shown as a negative control. The rAAV yield is shown as the amount of DNA-resistant particles (DRP) per cell. [Figure 2B] Figure 2B is a graph showing rAAV vector yields in large-scale vector production by triple transfection. An example of the overall titer obtained for a plurality of vector lots is shown. [Figure 2C] Figure 2C is a graph showing the in vitro performance of the AAV2 / HAS2 vector. 293 cells were infected at various MOIs, and the HA level in the conditioned medium was quantified after 3 days. [Figure 2D] Figure 2D is a graph showing the in vitro performance of the AAV5 / HAS2 vector. [Figure 3A] Figure 3A is a graph showing the change in body weight after intra-articular injection of the rAAV / HAS2 vector in normal canine joints. [Figure 3B] Figure 3B is a graph showing the cartilage score. [Figure 3C] Figure 3C is a graph showing the synovium score. [Figure 4A] Figure 4A is a schematic diagram showing the sample collection locations for rAAV vector quantification in canine synovium. [Figure 4B] Figure 4B is a graph showing the quantification of vector genome copy number in synovial sample #3. [Figure 4C] Figure 4C is a graph showing the vector genome copy number of synovial sample #1. The vector doses of AAV2 and AAV5 are shown as follows: L = low, M = medium, and H = high (similar to Figures 3A - C). All tissue samples were collected 28 days after rAAV vector delivery, and BGHpA was analyzed by qPCR. [Figure 5A]Figure 5A is a graph showing vector-derived cHAS expression in synovial sample #3. [Figure 5B] Figure 5B is a graph showing the vector mRNA in synovial sample #1. [Figure 5C] Figure 5C is a graph showing the vector genomes and vector-derived mRNAs of individual dogs analyzed using synovial sample #3. The vector doses for AAV2 and AAV5 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] Figure 6A is a schematic diagram showing the locations of femoral condyle and tibial plateau samples collected to detect cartilage rAAV vectors and mRNA. [Figure 6B] Figure 6B is a graph showing the vector genomes and vector-derived mRNAs of individual dogs analyzed using femoral condyle sample #1. In addition, the vector genome copy number on the opposite side (the right joint that was not injected) is shown (except for sample #22, which was not tested). [Figure 6C] Figure 6C is a graph showing the mean vector genomes (injected and non-injected joints) and mRNA copies for each group. The vector doses for AAV2 and AAV5 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] Figure 6D is a graph showing the average vector genome (PBS or vector-injected joint) and mRNA copies in the tibial plateau for each group. [Figure 7A] Figure 7A is a graph showing the vector genomes of synovial tissue (samples #3 and #1) and cartilage (femoral condyle and tibial plateau) in each treatment group of tissue collected from the left posterior knee joint. The values ​​shown in Figures 7A and 7B represent the mean ± standard deviation of the group (n=5 / group). [Figure 7B]Figure 7B is a graph showing the quantification of vector genomes and mRNA derived from the rAAV5 / HAS2 vector in various tissues. [Figure 8A] Figure 8A is a graph showing HA levels in canine synovial fluid. HA levels were quantified in SF samples collected on day -7 (baseline) and day 28. Each animal's HA level was normalized relative to the baseline level and expressed as a percentage of HA at day 28 compared to the week before vector administration. [Figure 8B] Figure 8B is a graph showing the HA levels in canine synovial fluid at day 7 (control line) and day 28. The arrows indicate animals with higher HA levels at day 28 compared to the control line (before treatment). [Figure 9] Figure 9 shows the complete amino acid sequence of inulubrisin. The framed region indicates the location of exon 6 (mucin domain). Underlined amino acids (378 to 782) are deleted in the shortened rubrisin (hereafter referred to as "cLub1" or "cLub1co"), and the locations of KEPAPTT-like repeats (potential O-linked glycosylation sites) are shown in bold. When a sequence name ends in "co", it means that the DNA sequence is codon-optimized. Similarly, "nonco" means that it is not codon-optimized. [Figure 10] Figure 10 is a schematic diagram showing the plasmids constructed and used in the experiment. The plasmids include shortening (i.e., internal deletion), a codon-optimized inulubrisin sequence (cLub1co), a promoter (minCBA or CBA), and a BGHpA site. Some constructs include modifications in which an N- or C-terminal His tag (C-term) and an ATG (latent start codon) are removed from the intron sequence. The previral AAV rubrisin also includes flanking ITR sequences at both ends. [Figure 11A] Figure 11A is a graph showing the mRNA copies / cell generated when 293 cells were transfected with minCBA cLub1, CBA CLUB1, and CBH cLub-nonco. [Figure 11B]Figure 11B is a graph showing the mRNA copies / cell generated when 293 cells were transfected with ΔATG / 6His / N', 6His / N', 6His / C', WT cLub, and EGFP constructs. [Figure 12A] Figure 12A is an anti-rubricin Western blot showing secretory rubricin levels in concentrated medium (plasmid is as described above). Canine synovial filtrate was used as a positive control. [Figure 12B] Figure 12B is a Western blot showing rubrisin production derived from a previral rubrisin expression plasmid. Two clones were analyzed and compared with the expression obtained using the minCBA-cLubco plasmid. Untransfected culture medium and cells transfected with the EGFP expression plasmid were used as negative controls. [Figure 13A] Figure 13A is a graph showing vector yield in small-scale vector generation for an AAV2 vector encoding inulbricin. Two cLub clones (- / + 6xHis tag) were analyzed and compared to packaging of EGFP and HAS2 expression cassette-holding previral (ITR-containing) plasmids. Negative controls included untransfected cells and transfections lacking the AAV2 capsid expression plasmid. [Figure 13B] Figure 13B is a graph showing vector yield in small-scale vector production for an AAV5 vector encoding inulbricin. Previral plasmids for EGFP and cLub expression cassettes were transfected together with the AAV5 capsid expression plasmid. [Figure 14] Figure 14 shows an anti-rubricin Western blot illustrating the in vitro expression of inulbricin derived from the rAAV5 vector. 293 human cells were infected with varying amounts of rAAV5 / minCBA-cLub1 for 72 hours, followed by concentration of conditioned culture media. Culture media of AAV5 / CBA-EGFP-infected cells were used as a negative control. Culture media of cells transfected with a previral rubrisin expression plasmid were used as a positive control. [Figure 15] Figure 15 is a table summarizing the sequence numbers. [Figure 16-1] Figure 16 shows the alignment of canine and human lubricin. [Figure 16-2] Figure 16-1 continued. [Figure 17] Figure 17 is a graph showing HA levels in canine synovial fluid at various time points using the MMR model. Synovial fluid was collected one week before OA induction (pre), two weeks after induction, and before the joint administration test (day 0) and 57, 112, and 182 days after the joint delivery test. [Figure 18A] Figure 18A is a graph showing the detection and expression of the rAAV5 vector 182 days after vector administration in synovial samples from canine OA joints. [Figure 18B] Figure 18B is a graph showing rAAV5 vector detection and expression in canine OA joint cartilage (femoral condyle) 182 days after vector administration. [Figure 18C] Figure 18C summarizes the detection of rAAV5 vector genome and cHAS2 mRNA in synovial and cartilage samples at day 182 in a canine MMR OA model. [Figure 19] Figure 19 shows, as an example, safranin-O stained sections of cartilage surfaces obtained from the medial side of joints in PBS- and two rAAV5 / cHAS2-treated canines. [Modes for carrying out the invention]

[0011] Detailed description of the invention Osteoarthritis (OA) is one of the most common causes of dysplasia in mammals and dogs, affecting approximately 20% of dogs over one year of age. OA is a progressive, degenerative disease that results in pain, inflammation, and reduced joint mobility. Novel, safe, and effective treatments that improve joint lubrication and reduce inflammation and pain are needed for the management of OA. As disclosed herein, the applicants have found that recombinant adeno-associated virus (AAV) vectors can be used to deliver the gene encoding the therapeutic substance by a single intra-articular injection, with the goal of providing localized and continuous production of the substance in the joint. The rAAV vector was constructed using AAV2 and AAV5 capsid serotypes and encoded canine codon-optimized hyaluronic acid (HA) synthase-2 (HAS2). 22 healthy adult dogs (negative for AAV2 and AAV5 capsid serotypes) were given rAAV2 (1.5 and 10x10) 11 vg / joint), rAAV5 (5x10 11 Dogs were administered either vg / 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, while no significant changes were observed in the rAAV2-treated group. Vector genome (VG) was detected in the synovial fluid and most cartilage samples of all rAAV-treated joints. The rAAV5 vector 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 tested joints. In summary, our study demonstrated gene transfer and an acceptable safety profile to canine joint tissues using rAAV2 and rAAV5 vectors encoding HAS2 when administered with a single intra-articular injection in a limited number of dogs.

[0012] InuHA Synthase 2In a feature of the present 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 the uptake of the vector into articular cells (followed by transport into the cell nucleus) and result in the expression of a therapeutic substance. In some embodiments, the DNA genome comprises one or more AAV reverse terminal repeats (ITRs) that flank into one or more expression cassettes for in vivo expression of the therapeutic gene in an animal host. In some embodiments of the present invention, the viral gene will be absent or not expressed from the rAAV genome. In some embodiments of the present invention, once rAAV is administered to an animal and taken up by the animal's cells, the rAAV genome will persist as an extrachromosomal episome. In some embodiments, the rAAV of the present disclosure can persist in articular cells for a long period, for example, longer 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 (but not limited to these). 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. Selected transgene products can promote joint health by increasing joint lubrication and reducing pain and cartilage degradation.

[0013] In another feature of the present invention, the disclosure provides a method for treating an animal in need, the method comprising the step of administering a therapeutically effective amount of rAAV of the disclosure to the animal. In some embodiments, the method comprises the step of administering the proposed product directly to the affected joint of a dog. In some embodiments, only one treatment is sufficient to have an effect on a significant improvement in the animal's symptoms. In other embodiments, the treatment is repeated. In some embodiments, the treatment is repeated within 2-3 weeks of the first dose, and in other embodiments, the second dose is given more than 3 weeks after the first dose. In some related embodiments, the second dose includes administration of an rAAV having the same therapeutic gene, and the rAAV contains the same serotype capsid as the first treatment. In other related embodiments, the second dose includes administration of an rAAV having the same therapeutic gene, but the rAAV contains a different serotype capsid than the first dose. In some embodiments, the rAAV has a serotype 5 capsid. In related embodiments (where repeated doses are desired), the first dose may include administration of an rAAV having a serotype 5 capsid, and the second dose may include administration of an rAAV having a serotype 5 capsid. In several features, overexpression of the HAS2 protein in osteoarthritis joints increases HA levels in synovial fluid, promoting joint health by enhancing HA's lubricating, anti-inflammatory, and pain-relieving properties. HAS2 overexpression has been shown in vitro to result in elevated HA levels in culture medium across various cell types. This was demonstrated using CHO, 293, and COS cells, either as stable or transient transfection. To provide in vivo overexpression of HA in joints, HAS2 cDNA can be delivered to cartilage and / or synovial membrane (normal HA synthesis sites) using an rAAV vector encoding a HAS2 expression cassette. While not bound by either theory, gene introduction into cells would provide the HAS2 expression cassette with sustained HAS2 expression and subsequent HA production. Since the therapeutic vector would contain a ubiquitous promoter, it would not be subject to downregulation by inflammatory mediators present in osteoarthritis joints, unlike the endogenous HAS2 promoter. When the vector is administered by intra-articular injection, it can induce transduction of various cell types (the primary cell type being synovial cells). Ultimately, it was shown that HAS2 alone is sufficient for HA synthesis, and other associated proteins or components are considered unnecessary for in vitro HA production (Yoshida et al.).

[0014] InulbricinIn several features, rubrisin production in osteoarthritis joints is increased by intra-articular delivery of recombinant adeno-associated virus (rAAV) vectors encoding rubrisin as a potential treatment for canine osteoarthritis (OA). Rubrisin is a large secreted glycoprotein that functions as a lubricant and protects the cartilaginous surface of joints. This specification describes the discovery and construction of the full-length cDNA of inulubrisin. Using the said cDNA, a shortened codon-optimized version of inulubrisin (cLub1co) was designed. Subsequently, various rubrisin expression plasmids were constructed using the latter (shortened codon-optimized version). After transfection into HEK293 cells, these plasmids were characterized for rubrisin mRNA and protein production. The data clearly demonstrated the production of both rubrisin mRNA and secreted rubrisin derived from each construct. rAAV vectors were constructed using the cLub1co expression cassette, demonstrating the feasibility of rAAV / cLub1 vector construction. This synthetic construct was used to infect HEK293 cells, causing them to secrete inulbricin. The methods and compositions described herein can also be used for the therapeutic treatment of osteoarthritis. The terms “treatment” or “therapeutic treatment” refer, when they relate to osteoarthritis, and further when they are used herein and in the field of veterinary medicine, to the treatment, support for treatment, and / or acceleration of treatment of subject animals that already have osteoarthritis or are recovering from osteoarthritis (e.g., in the recovery phase), or to treatment aimed at slowing and / or reversing chondrotaxy in subject animals diagnosed with osteoarthritis or at risk of developing osteoarthritis. A key objective of treatment is to reduce the risk of developing chondrotaxy and ossification. As used herein, a subject animal is said to have osteoarthritis or be at risk of developing osteoarthritis if it is reasonably expected that the subject animal will develop progressive chondrotaxy associated with osteoarthritis. Whether an individual subject animal has osteoarthritis or is at risk of developing osteoarthritis can be readily determined by a practitioner of the corresponding field of veterinary medicine or medicine.

[0015] The methods and compositions described herein can also be used for the prophylactic treatment of osteoarthritis. The terms “prevention, prophylaxsis” and “preventative treatment, prophylactic treatment” refer to the treatment of healthy animals or animals suffering from unrelated diseases (provided that such animals are considered to be at risk of developing osteoarthritis) when they relate to osteoarthritis, and further when they are used herein and in the fields of human and veterinary medicine. The therapies and prophylactic treatments for osteoarthritis described herein utilize pharmaceutical compositions comprising vectors capable of expressing HAS or lubricin polypeptide in vivo, as well as methods and compositions for reducing or eliminating cartilage loss by inducing a sustained increase in hyaluronic acid or lubricin concentration in the joints. As used herein, a pharmaceutical composition is said to have “therapeutic efficacy” or be “therapeutically effective” if the administration of a certain amount is sufficient to cause a significant improvement in the clinical signs or measurable markers of osteoarthritis in a target mammal suffering from the disease. As used herein, a pharmaceutical composition is said to have “prophylactic efficacy” or be “effective” if the administration of a certain amount is sufficient to prevent the onset of osteoarthritis in a target animal. The invention also relates to vectors capable of in vivo expression in a host of HAS or rubrisin, or the aforementioned variants, fragments, or combinations thereof. In several embodiments, the HAS or rubrisin polypeptides used in the invention are generally suitable for the target species of interest (for example, a vector encoding canine HAS2 is delivered to a dog suffering from OA).

[0016] Examples of “variants” and “derivatives” described herein include (but are not limited to) HAS and rubrisin varieties and derivatives, which are not strictly identical to the nucleotide sequences disclosed herein, but are encoded by nucleotide sequences in which changes in the nucleotide sequence do not alter the encoded amino acid sequence, or such changes result in conservative substitutions of amino acid residues, deletions or additions of one or more amino acids, or substitutions of amino acid residues by amino acid analogs that do not significantly affect the properties of the encoded polypeptide (for example, the varieties or derivatives have activity exceeding approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the desired activity of the wild-type polypeptide). 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, modifications, additions, deletions, and derivatives that result in functional HAS or rubrisin derivatives are also described herein, and those skilled in the art will readily know how to prepare, certify, or select such variants or derivatives, and further how to test the HAS or rubrisin activity of such variants or derivatives. Those skilled in the art can optimize the expression of the HAS or rubrisin polypeptide of the present invention. For example, removal of hidden splice sites, adaptation of codon usage frequency by introducing a Kozak consensus sequence before the start codon, modification of codon usage frequency (but not limited to the foregoing), or combinations thereof improve expression.

[0017] The vector used in the present invention may include a nucleic acid sequence encoding a canine HAS2 polypeptide comprising the amino acid sequence shown 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 with SEQ ID NO: 2. The vector used in the present invention may include an inurbrisin polypeptide comprising the amino acid sequence set shown in SEQ ID NO: 7. In some embodiments, the inurbrisin polypeptide is an inurbrisin 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 with SEQ ID NO: 7. Sequence identity or homology can be determined by comparing sequences that have been aligned to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using one of several mathematical algorithms. A non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm by 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 sequence comparison is the algorithm by Myers & Miller (Myers & Miller, CABIOS 1988, 4, 11-17). Such algorithms are incorporated into the ALIGN program (version 2.0) (the ALIGN program is part of the GCG sequence alignment software package). When using the ALIGN program for amino acid sequence comparison, the PAM120 weight residue table, gap length penalty, 12, and gap penalty, 4 can be used. Yet another useful algorithm for identifying regions for 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, amino acid sequence comparison can be achieved by aligning the amino acid sequence of a polypeptide with a known structure to the amino acid sequence of a polypeptide with an unknown structure. Subsequently, the amino acids in the sequences are compared, and homologous amino acid groups are grouped together. This method detects conserved regions of polypeptides and reveals amino acid insertions and deletions. Homology between amino acid sequences can be determined using commercially available algorithms (see also the description of homology above). In addition to those specifically mentioned herein, BLAST, Gap-Adding BLAST, BLASRN, BLASTP, and the PSI-BLAST program (provided by the National Center for Biotechnology Information) should also be mentioned. These programs are widely used in the industry for this purpose and can align homologous regions of two amino acid sequences. In all of these search programs, the gap-adding alignment routine is essential for the database search itself. Gap addition can be stopped if desired. The initial penalty (Q) for gaps of length 1 is Q=1 for proteins and BLASTP, and Q=10 for BLASTN, but can be changed to any integer. The initial one-residue penalty (R) for extending gaps is R=2 for proteins and BLASTP, and R=10 for BLASTN, but can be changed to any integer. Sequences can be aligned to maximize overlap and identity while minimizing sequence gaps by any combination of Q and R values. The initial 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 and refer to amino acid residue polymers of any length. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, which includes deoxyribonucleotides or ribonucleotides. As used herein, the term “vector” refers to recombinant DNA or RNA plasmid or virus, which includes heterologous polynucleotides to be delivered to target cells, for example, in vivo. The heterologous polynucleotides may include sequences of interest for therapeutic purposes and may be in the form of expression cassettes. As used herein, the “vector” does not need to have replication capacity in the final target cells or target animals. As used herein, the term “recombinant” means a polynucleotide of semi-synthetic or synthetic origin, which is linked to another polynucleotide in a configuration that does not exist in nature or is not found in nature. As used herein, the term “heterogeneous” means that an entity originates from an entity that is genetically distinct from the rest of the entity being compared. For example, a polynucleotide can be placed in a plasmid or vector derived from a different source by genetic engineering, and so that polynucleotide is a heterogeneous polynucleotide. A promoter that is extracted from its original coding sequence and ligated to operate on a coding sequence other than the original sequence is therefore a heterogeneous promoter. The polynucleotides used in accordance with the present invention may include, for example, additional coding sequences within the same transcription unit, regulatory elements (e.g., promoters), ribosome binding sites, transcription termination factors, polyadenylation sites, additional transcription units under the control of the same or different promoters, additional sequences such as sequences that enable host cell cloning, expression, homologous recombination, and any constructs that may be desired to provide embodiments of the present invention.

[0020] In some features, the disclosure provides a method for treating a subject mammal suffering from or at risk of developing osteoarthritis, the method comprising administering to the subject mammal a therapeutically effective amount of adeno-associated virus (AAV) comprising a nucleic acid sequence encoding a bone-protective or bone-regenerative polypeptide and optionally linked to a promoter, wherein the polypeptide is expressed in vivo in an amount effective to alleviate or prevent symptoms of OA in the subject mammal. In some embodiments, administration is via 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), the biologically active cleavage forms thereof, or combinations thereof. In some embodiments, the target mammal may be human, dog, or cat. In a specific embodiment, the target animal is a dog. In some embodiments, the target mammals suffer from or are at risk of developing chronic osteoarthritis.

[0021] In other embodiments, the polypeptide is canine HAS2 or canine rubrisin. In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide has a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubrisin polypeptide has a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 6. In some embodiments, the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the HAS2 polypeptide has an amino acid sequence selected from polypeptides, fragments, variants, and homologues (each exhibiting in vivo HAS activity in the target animal) that are at least 90% identical to the sequence shown in SEQ ID NO: 2. "HAS activity" means the production of biologically active hyaluronic acid. In some embodiments, the AAV vector includes 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 rubrisin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7. In other embodiments, the rubrisin polypeptide has an amino acid sequence selected from polypeptides, fragments, variants, and homologues (each exhibiting in vivo rubrisin activity in the target animal) having at least 90% identity with the sequence shown in SEQ ID NO: 7. "Lubrishin activity" means providing lubrication in substantially the same manner and to substantially the same degree as endogenously produced rubrisin. Such lubricating activity can be measured according to techniques known in the art (see, for example, 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 the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase (CK) gene promoter. In yet another embodiment, the AAV includes an AAV2 or AAV5 capsid.

[0022] In another feature, the present disclosure provides a method for increasing hyaluronic acid production in both chondrocytes and / or synovial cells of mammals (e.g., humans or dogs). In one embodiment, the method may include the steps of administering recombinant AAV ("rAAV") comprising an rAAV vector genome (containing nucleic acid encoding HAS2) to a mammal (e.g., a human or a dog), allowing sufficient time for the HAS2 enzyme to be expressed and subsequently catalyze the production of additional hyaluronic acid, thereby increasing the hyaluronic acid levels of the mammal. This disclosure also provides a method for increasing the production of rubrisin polypeptide in both chondrocytes and / or synovial cells of mammals (e.g., humans or dogs). In one embodiment, the method may include the steps of administering an rAAV comprising an rAAV vector genome (containing nucleic acids encoding rubrisin) to a mammal (e.g., a human or a dog), allowing sufficient time for rubrisin to be expressed, thereby increasing the rubrisin levels in the dog. In one embodiment, HAS2 is produced in sufficient quantities after administration of rAAV containing the nucleic acid encoding HAS2, to treat or prevent symptoms of OA in mammals (e.g., humans or dogs). In another embodiment, rubrisin is produced in sufficient quantities after administration of rAAV containing the nucleic acid encoding rubrisin to treat or prevent symptoms of OA in mammals (e.g., humans or dogs). In one embodiment, HA levels are restored to levels found in healthy mammals (e.g., humans or dogs). 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, rubrisin levels are restored to levels found in healthy mammals (e.g., humans or dogs).

[0023] In another feature, the Disclosure provides a method for treating dogs that have or are at risk of developing OA, the method comprising administering to the dogs a therapeutically effective amount of an AAV vector containing a nucleic acid sequence encoding HAS2 or a rubrisin polypeptide, which is ligated to act on a promoter. In another embodiment, the Disclosure provides a method for treating humans that have or are at risk of developing OA, the method comprising administering to the humans a therapeutically effective amount of an AAV vector containing a nucleic acid sequence encoding HAS2 or a rubrisin polypeptide, which is ligated to act on a promoter. In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubrisin polypeptide is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 6. In some embodiments, the AAV encodes a HAS2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the AAV encodes a rubrisin polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7, or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7. In any embodiment, the promoter can be selected from the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, β-actin gene promoter, CBA promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter.

[0024] In another feature, the disclosure provides a method for preventing the development of OA in a target mammal at risk thereof, comprising the step of administering a therapeutically effective amount of rAAV to a dog, comprising an rAAV vector genome comprising a nucleic acid sequence encoding HAS2 or a rubrisin polypeptide, which is linked to a promoter. In one embodiment, the nucleic acid sequence encoding the HAS2 polypeptide is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubrisin polypeptide is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 6. In another embodiment, the nucleic acid encodes a HAS2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or the nucleic acid encodes a rubrisin polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the promoter can be selected from the group consisting of the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter. In some embodiments, the rAAV vector contains CBA-cHAS2co-BGH. In other embodiments, the rAAV vector contains 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 rubrisin polypeptide, which is ligated to actuate a promoter. In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubrisin polypeptide is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 6. In some embodiments, the nucleic acid encodes a HAS2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or the nucleic acid encodes a rubrisin polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7. In certain features, the present disclosure provides a pharmaceutical composition comprising a recombinant viral vector encoding and expressing HAS or rubrisin in vivo in a mammalian host, and optionally one or more pharmaceutically acceptable carriers, excipients, or vehicles. In another embodiment, the Disclosure provides a method for treating a target mammal that has or is at risk of developing osteoarthritis, the method comprising the step of intra-articular administration of a therapeutically effective amount of the pharmaceutical composition detailed above to the target mammal. In one embodiment, the target animal is a human or a dog. In another feature, the disclosure provides adeno-associated virus (AAV)-based biological delivery and expression systems for use in the treatment or prevention of joint osteoarthritis in humans or mammals. In some embodiments, the method is achieved by long-term gene expression of human or mammalian HAS2 or rubrisin in synovial cells and / or chondrocytes, followed by delivery of rAAV, the rAAV comprising a nucleic acid sequence encoding human or mammalian HAS2 or rubrisin, left and right AAV reverse terminal repeats (L ITR and R ITR), an AAV packaging signal, and optionally a nonviral, non-coding stuffer nucleic acid sequence. In some embodiments, the expression of the human or mammalian HAS2 or rubrisin gene in synovial cells and / or chondrocytes is regulated by an inducible inflammation promoter. The promoter is located upstream of the reading frame of the nucleic acid sequence encoding human or mammalian HAS2 or rubrisin and is specifically activated by an increase in the level of an immunostimulant.

[0026] In some embodiments, the inflammation-inducing promoter is selected from: the NF-KB promoter, the interleukin-6 (IL-6) promoter, the interleukin-1 (IL-1) promoter, the tumor necrosis factor (TNF) promoter, the cyclooxygenase-2 (COX-2) promoter, the complement factor-3 (C3) promoter, the serum amyloid A3 (SAA3) promoter, the macrophage inflammatory protein-1a (MIP-1a) promoter, and the hybrid constructs thereof. In some embodiments, the rAAV vector genome comprises the nucleic acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 6, or the aforementioned bioeffective variants. In some embodiments, the AAV system comprises nucleic acids encoding marker genes that enable monitoring of the vector genome in synovial and chondrocytes. In some embodiments, the vector comprises the nucleic acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 6, or the aforementioned conserved sequences encoding the same amino acids. In some embodiments, the rAAV vector genome comprises nucleic acids encoding the HAS2 polypeptide shown in SEQ ID NO: 2 or the rubrisin polypeptide shown in SEQ ID NO: 7. The rAAV vector genome includes a nucleic acid molecule having at least 80% or 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 3. In other embodiments, the rAAV vector genome includes a nucleic acid molecule having at least 80% or 90% sequence identity with the nucleic acid sequence shown 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 rubrisin, left and right AAV reverse terminal repeats (L ITR and R ITR), a packaging signal, and optionally a nonviral, non-coding stuffer nucleic acid sequence, and for the treatment or prevention of osteoarthritis (OA), the expression of the human or mammalian HAS2 or rubrisin gene in synovial cells and / or chondrocytes is regulated by an inducible inflammation promoter and specifically activated by an increase in the level of immunostimulants.

[0027] Virus particles and methods for generating virus particlesFurthermore, provided herein are viral particles containing nucleic acids encoding HAS2 or rubrisin. Viral vectors can be used to deliver HAS2 or rubrisin-encoding nucleic acids to express the protein in target cells at a specific location (e.g., joints). Many viral species are known, and many have been studied for the purpose of delivering nucleic acids to target cells. Exogenous nucleic acids can be inserted into vectors (e.g., adeno-associated viruses (AAV)). In some embodiments, the viral particle is a recombinant AAV particle comprising a nucleic acid containing one or two AAV ITRs and a sequence encoding HAS2 or rubrisin as described herein, which is flanked by one or two ITRs. The nucleic acid is encapsulated within the AAV particle. The AAV particle also contains a capsid protein. In some embodiments, the nucleic acid comprises components linked in the direction of transcription to enable operation, a control sequence (including transcription start and end sequences), and a target protein-coding sequence (e.g., a nucleic acid encoding a fusion protein). These components are flanked at the 5' and 3' ends by functional AAV ITR sequences. A “functional AAV ITR” means that the ITR functions as intended for the 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 (both of which are incorporated herein by reference in their entirety)). To carry out some features of the present invention, the recombinant vector contains all of the AAV sequence essential for at least encapsulation and the physical structure for infection by rAAV. The AAV ITR used in the vector of the present invention does not need to have a wild-type nucleotide sequence (e.g., the one described by Kotin (Kotin, Hum. Gene Ther., 1994, 5:793-801)), but may be modified by nucleotide insertion, deletion, or substitution, or may be derived from any of several AAV serotypes. More than 40 AAV serotypes are known to date, and novel serotypes and variants of existing serotypes continue to be identified. See, for example, 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 to be within the scope of the present invention.In some embodiments, the rAAV vector is a vector derived from AAV serotypes 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 includes the ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In yet another embodiment, the rAAV particle includes the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10.

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

[0029] In some features, a method is provided for generating any rAAV particles disclosed herein, the method comprising the steps of: culturing a host cell under conditions for the generation of rAAV particles (wherein the host cell comprises (i) one or more AAV packaging genes (each AAV packaging gene encoding an AAV replication or encapsulation protein), (ii) an rAAV probe vector comprising nucleic acids encoding any fusion protein disclosed herein that are flanked by at least one AAV ITR, and (iii) an AAV helper function); and (b) recovering the rAAV particles generated 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 encapsulating 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, “purified” includes rAAV particle preparations that lack at least some of the other components that may also be present where the rAAV particles naturally exist or where the rAAV particles were initially prepared. Thus, isolated rAAV particles, for example, can be prepared using purification techniques to concentrate the particles from a source mixture (e.g., culture lysate or production culture supernatant). Concentration can be measured in various ways, for example, by the proportion of DNase-resistant particles (DRPs) present in the solution or by infectivity. The same can also be measured by comparison with a second potential interfering substance (e.g., impurities) present in the source mixture. The aforementioned contaminants include contaminants from production culture or contaminants from processing (including helper viruses, culture medium components, etc.). Furthermore, provided herein are pharmaceutical compositions comprising rAAV particles containing the nucleic acid encoding HAS2 or rubrisin of the present invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions may be suitable for various administration methods described herein (e.g., systemic or topical administration). The pharmaceutical compositions of rAAV containing the nucleic acid encoding HAS2 or rubrisin described herein can be administered systemically, for example, by intravenous injection, by catheter (see U.S. Patent No. 5,328,470), or by stereotactic injection (Chen et al., 1994, PNAS, 91: 3054-3057). In some embodiments, the compositions comprising rAAV and a pharmaceutically acceptable carrier described herein are suitable for administration to humans. Such pharmaceutically acceptable carriers may be sterile liquids, such as water and oils (including oils of petroleum, animal, plant, or synthetic origin), such as peanut oil, soybean oil, or mineral oil. Saline solutions, dextrose water, polyethylene glycol (PEG), and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions may further contain additional components, such as preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, and thickeners. The pharmaceutical compositions described herein may be packaged in single-unit or multi-unit dosing forms. Compositions are generally formulated as sterile and substantially isotonic solutions.

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expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy.Molecular Therapy 18:1983-1994 Hyc A et al.Pro- and anti-inflammatory cytokines increase hyaluronan production by rat synovial membrane in vitro.Intern J Molec Medicine 2009, 24:579-585 Itano N et al.1999.Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.JBC 1999, 274:25086-92 Itano N et al.1999.Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.JBC 1999, 274:25086-92 Keiser NW et al., Engelhardt.2011.Unique characteristics of AAV1, 2, and 5 viral entry, intracellular trafficking and nuclear import define transduction efficiency in HeLa cells.Hum Gene Ther 22:1433-1444 Kwiecinski et al.2011: The effect of molecular weight on hyaluronan’s cartilage boundary lubricating ability-alone and in combination with proteoglycan 4.Osteoarthritis Cartilage 19:1356-62 Kyostio-Moore S et al.2015.Over-expression of cystatin C in 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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 prepared and packaged into AAV2 or AAV5 capsids. Large vector lots were prepared by triple transfection and purified by CsCl gradient. Vector yield was quantified by qPCR against bovine growth hormone (BGH) polyA site (pA). A total of four lots were prepared for AAV2 / HAS2 (three of which were pooled for in vivo testing, 2 x 10⁻⁶). 13 DRP / Total Quantity). For AAV5 / HAS2, there are 2 lots (5x10). 12 We prepared DRP (Damage Reduction Potential / Total Amount), tested the consistency of production yield, and obtained a sufficient amount of virus. 22 healthy adult dogs (negative for AAV2 and AAV5 capsid serotypes) were given rAAV2 (1, 5 and 10x10) 11 vg / joint), rAAV5 (5x1011 rAAV5 (vg / joint) or PBS (control) was administered by intra-articular injection. Histopathological analysis showed minimal synovial inflammation in rAAV5-treated joints, while no significant changes were observed in the rAAV2-treated group. Vector genome (VG) was detected in the synovium of all rAAV-treated joints, and also in the majority of cartilage samples. The rAAV5 vector resulted in higher VG detection and mRNA expression in both tissues compared to rAAV2. An increasing trend in HA levels in the synovial fluid of treated joints was observed. In summary, the disclosed results reveal a gene transfer and acceptable safety profile to canine joint tissues upon single intra-articular injection administration to dogs.

[0032] Example 1b - Method Cloning and construction of HA expression vectors The canine HAS2 gene (GenBank XM 539153.3 (SEQ ID NO: 1)) was codon-optimized for expression in dogs using the GeneArt / Invitrogen algorithm. This codon-optimized canine HAS2 cDNA (1656 bp (SEQ ID NO: 3)) was synthesized using a flanking NheI-NsiI restriction enzyme site. This fragment was then cloned into a plasmid containing a ubiquitous chicken β-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 the maxi kit (Qiagen). cHAS2 in vitro expression and HA production293 cells were transfected with a plasmid vector containing cHAS2, and after 3 days, the conditioned culture medium and cell lysates were collected in 250 μL of RIPA buffer + protease inhibitor. Cell lysates were rotated to remove cell debris, and 30 μL of cell lysates were loaded onto a 4-12% nu-PAGE gel and electrophoresed in 1x Mops buffer. This protein gel was transferred to a nitrocellulose membrane and tested for anti-HAS2 as a probe overnight at 4°C in PBS-T with 5% milk and 0.1% Tween-20. Donkey anti-goat secondary antibody (1:5000 dilution) was used as the secondary antibody. Beta-actin detection was used to indicate that the cell lysates were loaded in equal volumes. Quantification of HA levels and molecular weight in in vitro cultures HA production by HAS2-expressing cells was evaluated by transfecting 293 cells with :pCBA-HI-cHAS2-BGHpA (using Optimen or complete medium). HA levels in conditioned cultures were quantified using an HA test kit (Corgenix, Inc.). The kit contains HA-binding protein derived from agrecan. HA molecular weight was determined by electrophoresis of concentrated conditioned cultures on agarose gels. Various HA size markers were run in parallel (Select-HA HiLadder, Hyalose, Austin, TX). Similar gels were run in parallel and then digested with hyaluronidase for 24 hours. Both gels were stained with All-stain. Production of rAAV vectors containing cHAS The cHAS2 expression cassette was cloned into an AAV ITR-containing plasmid, and an expression cassette was created by flanking with an AAV reverse-end repeat (previral plasmid pDC627) to construct psITR / CBA-HI-cHAS2-BGHpA. 600 bp of stuffer DNA (chromosome 16 P1 clone 96.4B) was added upstream of the expression cassette to create a total viral vector genome of 4500 bp. To test the packaging of the cHAS2 expression cassette-containing plasmid, 293 cells were sampled in 8x10⁶ cells. 5Cells were seeded per well (6-well plate), and the following day they were transfected by duplicate with psITR / CBA-HI-cHAS2-BGHpA or psp70 / EGFP, pHLP-19cap2 or p5repCMVcap5 plasmid and pAdHELP (CaPO4 kit (Promega)). Cells were collected after 3 days, and the titer of lysates for BGHpA copies was measured using qPCR and BGHpA sequence (SEQ ID NO: 12-14) primers / probes. Plasmids containing BGHpA were used as standard materials. rAAV virus yield was expressed as the amount of DNase-resistant particles (DRPs) per cell. Large-scale vector generation was performed using triple transfection with psITR / CBA-HI-cHAS2-BGH, pIM45BD rep-cap plasmid for AAV2 vectors, and pHLP19-cap5 and pAdHELP for AAV5 vectors. The vectors were purified using CsCl, and the titers of the resulting vector lots were measured using TaqMan analysis and BGHpA sequence primers / probes (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 synovial cells and chondrocytes) was tested in rabbit cells. Cells were infected with 1e5 DRP / cell and cultured for 3 days. Cell lysates were collected and HAS2 protein was detected by Western blotting, and HA levels were further quantified as described above. To test the effects of HA on the production of matrix-degrading proteases, inflammatory cytokines, and cartilage structural protein synthesis under symptomatic conditions, cells were first infected with the rAAV vector, followed by IL-1β 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 Mixed-breed dogs (male and female, 8-10 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 intra-articular routes (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 (pain, limping, swelling of the injected joint and other abnormalities) once daily for 7 days before injection, twice daily for 7 days after injection, and once daily during the subsequent test period. Animals were sacrificed after 4 weeks. Whole blood samples were collected at -7, 1, 14, and 28 days post-vector administration for white blood cell (WBC) counting. Synovial fluid (SF) samples were collected at -7, 1, 14, and 28 days for HA level quantification. 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 evaluated for the severity of chondral lesions and proteoglycan loss (score 0 - 5). Since synovial hyperplasia was not observed, synovial lesion scores were determined by the density of inflammatory cells (score 0 - 5).

[0033] Example 1c - Results Codon optimization and fabrication of HAS2 expression cassettesMammalian 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 using GeneArt / Invitrogen. This resulted in a nucleotide sequence with 78% similarity to the original GenBank sequence. The GC content of the optimized cDNA increased from 44.4% to 59.0%. Using this cDNA, a ubiquitous expression plasmid with a CBA promoter was constructed, enabling constitutive expression of HAS2 different from that with the endogenous promoter (Figure 1A). The CBA promoter is less affected by various pro-inflammatory 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 cell lysates were subsequently analyzed for HAS2 protein (membrane protein) by Western blotting. Cells transfected with the expression plasmid showed a band of 64 kDa, the expected size of cHAS (data not shown). Next, we determined whether overexpression of HAS2 protein in 293 cells resulted in increased HA detection in the culture medium (showing both HA production and secretion of HA across the cell membrane). HA levels in the culture medium of cells transfected with pCBA-cHAS2 were increased 6.5-fold and 9-fold, respectively, compared to untransfected cells and CBA-EGFP transfected cells (Figure 1B). Therefore, these data confirmed that in vitro overexpression resulted in increased HA levels 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 culture medium obtained from cells transfected with HAS2 expression cassettes. The size of this substance was greater than 1.5 megadaltons (MDa) (based on estimation using HA molecular weight markers). This substance disappeared after hyaluronidase digestion, indicating that the substance was HA (Figure 1C). Construction of an rAAV vector containing a HAS2 expression cassette The cHAS2 co-expression cassette was subsequently cloned into a plasmid containing the AAV ITR. A schematic diagram of the resulting viral genome is shown in Figure 1A. The ability to generate rAAV vectors containing AAV2 and AAV5 capsids and HAS2 cDNA was tested in small-scale packaging experiments (Figure 2A), followed by large-scale vector generation. Both AAV2 and AAV5 vectors were successfully produced using a standard triple transfection method (Figure 2B). The ability of this material was tested by infection of 293 cells and analysis of HA production levels in the culture medium. Both AAV2 and AAV5 vectors resulted in a dose-response increase in HA in the culture medium (Figures 2C, D). Evaluation of rAAV / HAS2 vectors in the joints of normal dogsrAAV2 and rAAV5 vectors containing cHAS2 were delivered intra-articularly to the joints of normal dogs, and the animals were evaluated for 28 days. No adverse clinical signs, weight changes (Figure 3A), crawling, or death were observed during the study. -On day 7, some animals showed an increase in white blood cell (WBC) count, which may be due to transport stress. WBC counts on days 1, 14, and 28 were generally within the normal range. Histological evaluation of the knees on the PBS- and AAV-injected side (left) and the contralateral side (non-injected) showed very slight proteoglycan degeneration and cartilage degeneration (score range 0-0.5 (maximum score 5)). These minimal changes were typical age-appropriate spontaneous changes. Minimal synovial changes were observed in the PBS- and AAV2-treated joints as well as the contralateral joint (Figure 3C). Minimal to mild synovitis (generally extending to the joint capsule and medial collateral ligament) was observed in all left knees of male and female animals treated with the AAV5 vector (no synovitis was observed in the contralateral joint). Therefore, overall, this treatment was well-tolerated with few observed adverse effects. Tissue samples collected from synovial membrane and cartilage were analyzed for the detection of viral genomes (Figures 4A, 6A). Synovial samples collected closest to the injection site (sample #3) showed the presence of vector genomes in all AAV-treated joints (Figure 4B). AAV2-treated joints contained roughly 0.01 to 2 vector genomes (VG) / cell. Interestingly, a slight dose response was observed in AAV2 despite a 10-fold difference between the low-dose and high-dose groups. Joints treated with the AAV5 vector 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 pronounced in the low-AAV2-treated group and more sporadic in the high-AAV2-dose and AAV5-groups (data not presented). Synovial samples collected further above the injection site (sample #1) were analyzed to assess the diffusion of AAV into the joint (Figure 4C). Joints injected with low-dose AAV2 showed more consistent VG detection. These levels were similar to those measured in synovial sample #3. In the AAV5 treatment group, all synovial sample #1 consistently had detectable VG (within 3x). However, these were lower than the VG levels detected in synovial sample #3, thus revealing position-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 low, medium, and high-dose AAV2-treated groups, respectively, while all AAV5-treated joints had detectable mRNA copies (Figure 5A). Vector expression was also detected for the AAV5 vector in synovial sample #1, but its level was low, similar to the low VG detection at this site (Figure 5B). mRNA detection showed a good correlation with VG detection. The respective mRNA and VG DNA from individual injected joints in synovial sample #3 are shown as examples (Figure 5C).

[0034] Detection of vector genomes in canine cartilage Cartilage samples collected from the femoral condyle and tibial plateau were analyzed for viral genome detection (VG, Figure 6A). Vector DNA and mRNA detected in each individual injected joint, as well as group averages in the femoral condyle, are shown as examples (Figures 6B, C). This data indicates that the AAV5 vector is present in cartilage in a consistent manner and showed comparable levels of vector-derived transcripts. A similar dose of AAV2 vector (medium dose) produced similar VG levels to the AAV5 vector, but showed 100-fold lower mRNA levels. In addition, AAV2 VG copies appeared to have an inverse correlation with vector dose. rAAV5 injected joints also showed detection and expression of the vector in cartilage samples collected from the tibial plateau, but it was not detected at all in rAAV2 treated joints (Figure 6D). All vectors resulted in minimal detection of vector DNA in the contralateral (non-injected) joint. The results for synovial membrane and cartilage are summarized in Figure 7A. For gene transfer in the synovial membrane, the AAV5 vector produced nearly 10 times higher vector copies at the site of both synovial samples compared with the AAV2 vector. Gene transfer to cartilage was 10 to 20 times lower than that to the synovial membrane by AAV5, while the AAV2 vector genome was observed at similar levels in both synovial membrane and cartilage. The detection of rAAV5 vector-derived genome and mRNA is summarized in Figure 7B, showing consistent gene transfer and expression by the rAAV5 / HAS2 vector in all tissue samples tested. We consider these results to be highly unexpected. Analysis of HA levels in synovial fluid To determine whether any changes in synovial HA levels could be detected after rAAV vector administration, synovial HA levels were quantified in samples collected on day 7 (baseline) and day 28. Since high levels of variability were detected between animals, each animal's HA level was normalized to its baseline level. The data showed that both AAV2 / high dose and AAV5 / medium dose increased synovial HA levels on average compared to PBS-treated animals (Figures 8A and 8B).

[0035] Example 1d - Conclusion To provide in vivo overexpression of HA in the joints, the applicants constructed rAAV vectors using two capsid serotypes. The selection of AAV capsid serotypes is important because any neutralizing antibody already present in the target species can neutralize the therapeutic vector and thus prevent gene transfer by the rAAV vector. The results disclosed herein showed that the majority of dogs analyzed had low levels of neutralizing antibodies against both AAV2 and AAV5 capsids. Therefore, the applicants directly tested the localization of AAV2 and AAV5 capsids in target tissue (i.e., canine knee joints) after intra-articular injection. Since HA expression is expected to be beneficial to both synovial and chondrocytes, vector genome copies were quantified in canine synovial and cartilage samples, respectively. The data showed that AAV2 provided in vivo gene transfer to canine synovial and cartilage tissues with extremely inconsistent results, and furthermore, showed little dose-response, for reasons that are unclear. Similar experiments conducted in rabbit joints with osteoarthritis (OA) showed highly consistent detection of the rAAV2 vector genome using similar vector doses (Kyostio-Moore, 2015). In contrast to the AAV2 vector, the AAV5 vector genome was detected in a consistent manner across both tissue types (n=5 / group). Importantly, the detection of AAV5 in cartilage samples is surprising and unexpected. This is because cartilage has been reported to be difficult to transduce under in vivo conditions due to its extensive extracellular matrix, and to date, there are no other reports on the detection of AAV5 in cartilage of many animals after intra-articular delivery. In addition, the canine studies in this disclosure produced surprisingly high levels of the rAAV5 vector in canine synovial tissue, which was also approximately 2 log higher in the synovium compared to that in cartilage, indicating a preference for AVV5 in the canine synovial lining. This preferential expression pattern was also unpredictable prior to this disclosure. In addition to high levels of vector detection, mRNA analysis confirmed recombinant HAS2 expression in canine synovial and cartilage tissues, demonstrating that the CBA promoter is functional in both tissue types. Furthermore, detection of AAV5 transcripts in cartilage samples confirmed that chondrocytes are transduced by the vector rather than by a virus hidden within the extracellular matrix of the cartilage. For AAV2, similar levels of vector genome and transcripts were also observed in the inner synovial layer. However, the mRNA expression of the AAV2 vector was surprisingly almost 100 times lower than the detection of the corresponding vector genome in cartilage samples, suggesting that some of the vector persists outside the chondrocytes, possibly retained in the extracellular matrix. These significant differences may only be understood after the applicants have conducted significant unconventional experiments. Although the vector was administered to only one joint in each animal, the vector genome was sometimes detected in the contralateral, non-injected joint. This was mostly observed in synovial samples obtained from the AAV2-treated joint. However, none of the animals in which the vector genome was observed in the contralateral joint had any detectable HAS2 transcripts in those joints. In summary, the data disclosed herein demonstrate that AAV5 capsids provide favorable gene delivery to canine joints via intra-articular delivery. This is due to the low pre-existing humoral immunity to AAV5 in the animals and the ability to transduce into joint tissue after intra-articular injection. Intra-articular injection can provide gene delivery not only to the synovial lining but also to chondrocytes of the cartilage. Both Tissue types will benefit from the increased HA synthesis provided by the gene delivery compositions and methods of this disclosure. Specifically, synovial membranes will benefit from enhanced ability to provide lubrication with synovial fluid, and cartilage will benefit from enhanced matrix bonding and thus improved cartilage health as a scaffold. These results suggest that overexpression of HA by AAV-mediated HAS2 gene introduction into diseased sites will improve the pathology and pain of OA. Reduce This indicates that. [Examples]

[0036] Construction and evaluation of the rubrisin AAV vector. Example 2a - Overview Intra-articular injection of recombinant rubrisin has recently been shown to reduce cartilage degeneration in a rat OA model (Flannery, 2006). However, rubrisin administered intra-articular has a very short half-life in synovial fluid, and most of the protein is removed within 72 hours (Vugmeyster, 2011). Therefore, repeated intra-articular injections are required, which is time-consuming, laborious, stressful, and expensive. In contrast to HAS2 (see Example 1), rubrisin is encoded by a large cDNA and further contains multiple DNA repeats in its mucin-like domain, which makes compatibility with rAAV vectors and high-level expression difficult. To circumvent this problem, the applicants have created a truncated inulubrisin cDNA and optimized a small expression cassette to enhance rubrisin production. Importantly, prior to this disclosure, neither the full-length inulubrisin sequence nor the truncated form disclosed herein was known. In short, the applicants constructed a full-length inulbricin cDNA and subsequently designed a shortened codon-optimized inulbricin version (cLub1co) using it. They then constructed various rubrisin expression plasmids using the latter. These plasmids were characterized for rubrisin mRNA and protein production after transfection of HEK293 cells. Data showed that both rubrisin mRNA and secreted rubrisin were produced from each construct. Finally, the applicants constructed an rAAV vector containing a cLub1 expression cassette and demonstrated the feasibility of rAAV / cLub1 vector production. HEK293 cells infected with this construct synthesized and secreted inulbricin.

[0037] Example 2b - Method Cloning of inulbrycinSince full-length canine rubrisin cDNA does not exist in GenBank (incomplete sequence: GenBank no. ABD38836.1), complete canine cDNA was obtained from a custom-synthesized canine cartilage cDNA library. To achieve this, overlapping fragments were constructed using qPCR with various primers. Subsequently, a shortened form of inulubrisin (cLub1) was designed using the full-length cDNA (SEQ ID NO: 4) (similar to the short published version of human rubrisin (Flannery, 2009)). This shortened inulubrisin contained a deletion in the sequence encoding amino acids 378 to 782. This shortened rubrisin 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 the plasmid. The plasmid contained a CMV enhancer, a chicken β-actin promoter, a truncated hybrid intron (Hib) (minCBA), and a bovine growth hormone (BGH) polyadenylation site. E. coli Stable II cells were transformed with the ligation reaction and grown at 30°C to minimize DNA rearrangement. The resulting clones were analyzed by restriction enzyme analysis, and the cloning junction was analyzed by DNA sequencing. Additional constructs were prepared. These additional constructs contained 6x histidine (6xHis) and modifications to two “ATG” sequences present in the intron sequence. In vitro rubrisin expression was analyzed using the expression plasmid. Inurbricin expression analysisHEK293 cells were transfected with a rubrisin expression plasmid using lipofectamine 2000 (Invitrogen), and the cells were grown for 72 hours. To analyze rubrisin mRNA expression, cells were collected and transcript levels were measured by a real-time (RT) qPCR assay. BGA pA-specific primers / probes were used for the assay (7500 Real-Time PCR System; Applied Biosystems, Foster City, CA). For proteinogenesis analysis, the culture medium was collected and concentrated approximately 20 to 30 times (100k MWCO filter, Millipore). Samples were electrophoresed on 4-12% Bis-Tris gel or 3-8% Tris-acetic acid (NuPAGE; Thermo Fisher Scientific) SDS-PAGE gel (reduced) using MOPS or Tris-acetic acid buffer, respectively. Rubrisin was detected by Western blotting using a mouse anti-rubrisin antibody (9G3, Millipore) (Ai, 2015) and a goat anti-mouse HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) as a secondary antibody. Fabrication of AAV / cLub1co The cLub1 expression cassette was cloned with an AAV reverse terminal repeat (ITR)-containing plasmid to create a flanked expression cassette (previral plasmid pDC627) by the AAV ITR, and psITR / minCBA-HI-cLub1co-BGHpA was constructed. To test the packaging of the cLub1 expression cassette-containing plasmid, 293 cells were sampled in 8x10⁶ cells. 5Cells were seeded per well (6-well plate), and the following day, psITR / minCBA-HI-cLub1co-BGHpA, or psp70 / EGFP, pHLP-19cap2 (AAV2), or p5repCMVcap5 (AAV5) plasmids and pAdHELP (Promega CaPO4kit) were transfected to package the vector into the AAV2 or AAV5 capsid. Cells were collected after 3 days, and vector yield was quantified by qPCR assay (7500 Real-Time PCR System) of lysates. Standard curves were used for BGH pA sequence-specific primers / probes (Applied Biosystems / Life Technologies) and serially diluted linearized plasmid DNA (containing BGH pA). rAAV virus yield was expressed as the amount of DNase-resistant particles (DRP) per cell (Clark, 1999). Experimental-scale vector production was performed using triple transfection with psITR / minCBA-HI-cLub1co-BGH, pHLP19-cap5 (for the AAV5 vector), and pAdHELP. The vector was purified by a CsCl gradient, and the yield was quantified as described above (University of Massachusetts Medical School, Worcester, MA).

[0038] Example 2c - Results Production of short inulbricin The inurubrisin sequence found in GenBank is missing a large portion of exon 6 (coding 857 amino acids), so a full-length inurubrisin cDNA (4017 bp (without stop codon), SEQ ID NO: 4) was constructed. The full-length inurubrisin cDNA codes for a protein containing a total of 1339 amino acids (SEQ ID NO: 5, Figure 9) (slightly smaller than the 1404 amino acid human sequence). At the amino acid level, this inurubrisin has 79% identity with the human rubrisin sequence (SEQ ID NO: 11, Figure 16). Because the full-length inulbursin is too large to fit into rAAV vectors due to package limitations, a shortened version of inulbursin was created. This shortened inulbursin version ("Lub1") was created by deleting the sequence encoding amino acids 378 to 782 of the mucin-like domain, yielding a 2949 bp long cDNA (SEQ ID NO: 6) encoding amino acid 983 (SEQ ID NO: 7). Despite the large deletion of the mucin-like domain, nearly 10 KEPAPTT-like peptide repeats remained. Importantly, none of these are identical to the standard human repeat sequences, but (even if they were) those skilled in the art could not have predicted that delivery of this shortened canine Lub1 would be effective in treating OA. These repeats are considered important for lubrication properties because they are potential O-linked oligosaccharide binding sites. Codon optimization of this short rubrisin (Lub1co, SEQ ID NO: 6) increased the GC content from 44% to 60% and achieved 74% nucleotide similarity to the original canine DNA sequence. Subsequently, a plasmid expression cassette containing the minCBA promoter, cLub1co, and BGHpA was constructed using this short canine cDNA (Figure 10). Expression plasmids with a 6xHis- tag and modifications to the putative ATG nucleotide sequence within the intron region (minimizing the false translation start site) were also constructed. Inurbricin expression analysisExpression of cLub1co from the minimal CBA promoter (minCBA-cLub1co) plasmid was confirmed in vitro by increased mRNA levels in transfected 293 cells (Figure 11A). Activity of the minCBA-cLub1co construct with short introns was approximately three times lower than that of the full-length CBA-HI construct (CBA-cLub1co). Transcription was barely observed with plasmids containing full-length rubrisin and non-codon optimized constructs (CBH-cLubr). Transcript analysis was also performed for expression cassettes with various modifications (Figures 10, 11B). Expression of minCBA-Lub1co was comparable to that of EGFP and C-terminal 6xHis-tagged constructs. Deletion of two putative ATG codons present in the hybrid intron appeared to enhance expression levels by approximately twofold. Another morphological change observed in Lubico-transfected cells also suggested Lub1 expression. This is because these changes were not present in non-transfected cells or EGFP plasmid-transfected cells (data not presented). The production of canine Lub1 protein from diverse expression plasmids was tested by Western blotting with antibodies against rubrisin, and proteins of 250–380 kDa were shown in concentrated culture medium (Figure 12A). The expected size based on 1339 amino acids is approximately 160 kDa, but the larger size and broader pattern signal are likely due to glycosylation. Detection was hardly observed in non-transfected cells or EGFP plasmid-transfected cells. In addition, ΔATG modification appeared to increase rubrisin detection, as well as observations of elevated transcript levels derived from this construct. Protein expression was also confirmed from previral AAV plasmids, showing comparable protein detection (Figure 12B). In summary, these results clearly demonstrate that plasmids containing canine rubrisin expression cassettes express and secrete glycosylated rubrisin protein. Construction of an rAAV vector containing an inulbricin expression cassette.Having confirmed inulbricin expression in the plasmid vector, we then tested whether the expression cassette could be packaged into AAV2 and AAV5 capsid serotypes in small-scale packaging experiments (Figure 13A, B). The data showed similar inulbricin packaging efficacy for both AAV2 and AAV5 capsids as observed with the EGFP expression vector. Adding a 6xHis- tag did not change the rAAV vector yield. Approximately 5-fold lower levels of packaging were measured with the AAV2 vector containing the canine HAS2 expression cassette. Subsequently, we evaluated scale-up vector generation by performing experimental-scale AAV5 / minCBA-cLub1 production. The vector yield was comparable to that of standard AAV2 and AAV5 vectors containing EGFP as a transgene (data not shown). Next, we tested the AAV5 vector in vitro for rubrisin production and secretion in HEK293 cells. Western blotting analysis of conditioned cultures demonstrated dose-dependent detection of inurbricin (Figure 14). In summary, the data showed that an rAAV vector could be constructed using a shortened version of inurbricin, and that cells infected with this vector could mediate rubrisin synthesis and secretion into the culture medium.

[0039] Example 2d - Conclusion As described above, rubrisin as a transgene presents several challenges for rAAV construction. Firstly, the size of the rubrisin cDNA containing the required expression elements exceeds the packaging capacity of rAAV, thus requiring a shorter cDNA. Interestingly, compared to the human rubrisin amino acid sequence of the mucin-like domain, the complete KEPAPTT repeat is absent in the canine sequence (Figure 16). In the construction of recombinant rAAV vectors, any repeating DNA sequence can present challenges, as repeat sequences can reduce the stability and integrity of the viral genome by causing DNA deletions and rearrangements during virus generation. However, given that a comparable vector yield was obtained compared to a standard EGFP reporter vector, the disclosed (and surprising) results demonstrate that the construction of rAAV viruses containing and expressing a novel canine rubrisin sequence is feasible. Thus, this is the first report to elucidate a single rAAV vector strategy for rubrisin gene delivery. [Examples]

[0040] In vivo efficacy study of AAV-HAS2 in a medial meniscus ligament release (MMR) model. The objective of this study was to evaluate the efficacy of HA synthase 2 gene therapy using macroscopic observation and histology in a canine post-osteoarthritis (OA) knee joint model. Twelve intact male research breeding mixed-breed dogs (foxhound phenotype, approximately 20-23 kg) were anesthetized, and medial meniscal resection (MMR) of the right posterior knee joint was performed using arthroscopy (-14 days). Phosphate-buffered saline (PBS control) or DNase-resistant particles (DRP) of recombinant AAV5 containing canine hyaluronic acid synthase 2 (cHAS-2) (5x10 11 The drug was administered intra-articularly on day 0 (n=6 animals / group). Plasma was collected from all dogs on days 0 and 182 for biomarkers of arthritis. Right and left synovial fluid was collected from all PBS control and cHAS-2 treated groups on days 0, 56, 112, and 182 for analysis of HA levels. On day 182, the dogs were euthanized, and cartilage defects induced by posterior knee joint ligament detachment (indicated by India ink staining) were measured. Joint tissue was collected for histopathological examination using the standard techniques of the International Association for Office Alignment Research (OA Research Society International (OARSI)). Macroscopic and histological data were analyzed using Kruskal-Wallis with GraphPad Prism 6 statistical software. Total HA levels in the synovial fluid were measured, but no treatment-related differences in total HA levels were observed (Figure 17). Synovial and cartilage samples were collected from the treated joints on day 182 and analyzed for detection of the viral genome (Figure 18A). Vector-derived DNA and mRNA were detected in each synovial sample (Figure 18A) and most cartilage samples (Figure 18B) from each rAAV5 / cHAS-2 injected joint. The data are summarized in Figure 18C, showing the mean vector genome and mRNA in both tissue samples. There was no evidence of local or systemic toxicity closely related to intra-articular administration of HA synthase-2 gene therapy. Consistent cartilage structure preservation was observed with cHAS-2 treatment compared to PBS treatment. Reduction in lesion size and depth at both the medial femoral condyle and medial tibial plateau joint surfaces was more pronounced in the femoral condyles of 4 out of 6 rAAV5 / cHAS2 treated dogs. In Figure 19, histopathological scores based on Cook et al. (2001) are shown in the left inferior angle of the medial femoral condyle and medial tibial cartilage images (2x). Canine 994731 / PBS had extensive erosions extending toward the median zone and showed considerable proteoglycan loss on both cartilage surfaces. No cartilage protective effect was observed. Canine 993107 (treated with rAAV5 / cHAS2) had a shallow lesion in the superficial zone of the femoral cartilage, but overall, there was good protection of the cartilage residue and little proteoglycan loss. The tibial plateau lesion was deeper toward the median zone and showed mild proteoglycan loss. Since the underlying cartilage was relatively normal, cartilage protective effect was present in the femoral condyle. Canine 992879, treated with rAAV5 / cHAS2, showed some proteoglycan loss in the femoral cartilage, but the overall morphology was preserved. The tibial plateau had clearly defined focal erosions, but most of the cartilage was preserved. Therefore, since the lesions were small and mild in severity, some degree of cartilage protection was demonstrated. In particular, one rAAV5-treated animal in which the vector could not be detected in the cartilage sample also had the largest tibial plateau lesion area (dog 993107, Figure 19). Conversely, synovial and cartilage both One of the rAAV5-treated animals (dog 992879) in which the vector was detected (however, mRNA was not detected in the cartilage) had the best cartilage structure. Therefore, the presence of the rAAV5-HAS2 vector is closely associated with optimal cartilage structure, and the absence of this vector is closely associated with the largest tibial plateau lesion area. Thus, despite the variability in vector / mRNA detection, the rAAV5 vector expressing HAS2 appears to have produced the desired clinical outcomes. In summary, these results revealed consistent rAA5-mediated gene transfer into the synovial membrane and cartilage of canine OA joints, with sustained vector-derived expression confirmed for at least six months. Histological analysis showed a reduction in cartilage lesions and slowed disease progression in most cHAS-2-treated joints, while little difference was observed in total synovial fluid HA levels. The latter may indicate that local HA expression in cartilage and synovium possesses some disease-modifying properties without increasing total synovial fluid HA levels. Alternatively, changes in molecular weight 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 present invention is further represented by the following numbered items: 1. A method for treating a target mammal suffering from osteoarthritis (OA), comprising the step of administering a therapeutically effective amount of recombinant adeno-associated virus (rAAV) intraarticularly to the target mammal, wherein the recombinant adeno-associated virus comprises a nucleic acid encoding a bone-protective or bone-regenerative polypeptide linked to a promoter, and the polypeptide is expressed in vivo in an amount effective to alleviate the symptoms of OA in the target mammal. 2. The method according to item 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), the biologically active cleavage form of the above, or a combination thereof. 3. The method according to item 1 or 2, wherein the polypeptide is a HAS2 polypeptide. 4. The method described in any of items 1-3, wherein the target mammal is a human, dog, or cat. 5. The method described in any of items 1-4, wherein the target mammal is a dog. 6. The method described in 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 with the amino acid sequence shown in SEQ ID NO: 2, or the aforementioned fragment, variant, or homologue exhibiting HAS2 in vivo activity in the target animal. 8. The method according to any of items 5-7, wherein the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. 9. The method according to any of items 5-8, wherein the nucleic acid encoding the HAS2 polypeptide has a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 3. 10. The method according to any of items 5-9, comprising an rAAV vector genome containing the following elements from 5' to 3': 5'AAV reverse end repeat (ITR), stuffer nucleic acid, promoter, intron (IN), cHAS2 codon-optimized cDNA, polyadenylation signal (pA), and 3'AAV ITR. 11. The method according to item 10, wherein the promoter is a chicken verte-actin (CBA) promoter. 12. The method according to item 1 or 2, wherein the polypeptide is a rubrisin polypeptide. 13. The method according to item 12, wherein the rubrisin polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 7, or the aforementioned fragment, variant, or homologue exhibiting in vivo rubrisin activity in the target animal. 14. The method according to item 13, wherein the rubrisin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7. 15. The method according to item 13 or 14, wherein the nucleic acid encoding the rubrisin polypeptide has a nucleotide sequence that is at least 90% identical to the nucleotide shown in SEQ ID NO: 6. 16. The method described in any of items 13-14, comprising an rAAV vector genome encoded by plasmid pITR / minCBA-HI-cLub1co-BGH. 17. The method according to either item 1-9 or 12-16, wherein the promoter is selected from the group consisting of the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, β-actin gene promoter, CBA promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase (CK) gene promoter. 18. The method described in item 1, wherein the AAV includes an AAV2 or AAV5 capsid. 19. A method for increasing hyaluronic acid production in canine chondrocytes and / or synovial cells, comprising the step of administering rAAV to a dog, wherein the rAAV comprises an rAAV vector genome comprising a nucleic acid encoding the HAS2 enzyme ligated to a promoter, and after administration, the HAS2 enzyme is expressed to catalyze the production of additional hyaluronic acid, thereby increasing the level of hyaluronic acid (HA) in the dog. 20. The method described in item 19, wherein HAS2 is produced in an amount sufficient to treat the symptoms of canine OA. 21. The method described in item 20, wherein HA levels are restored to levels found in healthy dogs. 22. A method for treating a dog suffering from OA, comprising the step of administering a therapeutically effective amount of rAAV to the dog, wherein the rAAV comprises an AAV vector genome containing a nucleic acid encoding HAS2, which is ligated to a promoter. 23. A method for treating a person suffering from OA, comprising the step of administering a therapeutically effective amount of rAAV to the person, wherein the rAAV comprises an AAV vector containing a nucleic acid encoding HAS2, which is ligated to a promoter. 24. The method according to any of items 19-23, wherein the nucleic acid encoding HAS2 has at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3, or encodes HAS2 having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. 25. The method according to any of items 19-23, wherein HAS2 has the amino acid sequence shown in Sequence ID No. 2. 26. A method for increasing the production of rubrisin in canine chondrocytes and / or synovial cells, comprising the step of administering rAAV to a dog, wherein the rAAV comprises an rAAV vector genome containing a nucleic acid encoding rubrisin, which is ligated to a promoter, so that the rubrisin is expressed after administration, thereby increasing the level of rubrisin in the dog. 27. The method according to item 26, wherein lubricin is produced in an amount sufficient to treat the symptoms of canine OA. 28. The method described in item 26, wherein rubrisin levels are restored to levels found in healthy dogs. 29. A method for treating a dog suffering from OA, comprising the step of administering a therapeutically effective amount of rAAV to the dog, wherein the rAAV comprises an rAAV vector genome comprising a nucleic acid encoding rubrisin, which is ligated to a promoter. 30. A method for treating a person suffering from OA, comprising the step of administering a therapeutically effective amount of rAAV to the person, wherein the rAAV comprises an AAV vector genome containing a nucleic acid encoding rubrisin, which is ligated to a promoter. 31. The method according to any of items 26-30, wherein the nucleic acid encoding the rubrisin polypeptide is at least 90% identical to the sequence shown in SEQ ID NO: 6, or the nucleic acid encodes rubrisin having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7. 32. The method according to any of items 26-30, wherein rubrisin has the amino acid sequence shown in Sequence ID No. 7. 33. The method according to any one of items 19-32, wherein the promoter is selected from the group consisting of the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter. 34. The method described in any of items 19-25, comprising an rAAV vector genome encoded by plasmid Ps-AAV-ITR / CBA-cHAS2co-BGH. 35. The method described in any of items 26-32, comprising an rAAV vector genome encoded by plasmid Ps-AAV-ITR / minCBA-HI-cLub1co-BGH. 36. A method for preventing the onset of OA in a target mammal at risk thereof, comprising the step of administering a therapeutically effective amount of rAAV to a dog, wherein the method comprises an rAAV vector genome containing a nucleic acid encoding HAS2, which is linked to a promoter so that the rAAV can act. 37. The method according to item 36, wherein the nucleic acid encoding the HAS2 polypeptide is at least 90% identical to the sequence shown in SEQ ID NO: 2, or encodes HAS2 having an amino acid sequence that is 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 target mammal at risk thereof, comprising the step of administering a therapeutically effective amount of rAAV to a dog, wherein the method comprises an rAAV vector genome containing a nucleic acid encoding rubrisin, which is linked to a promoter so that the rAAV can act. 40. The method according to item 39, wherein the nucleic acid encoding rubrisin is at least 90% identical to the sequence shown in SEQ ID NO: 6, or the nucleic acid encodes rubrisin having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7. 41. The method according to item 40, wherein the rubrisin 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 the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter. 43. The method described in item 26, comprising an rAAV vector genome encoded by plasmid Ps-AAV-ITR / CBA-cHAS2co-BGH. 44. The method described in item 26, comprising an rAAV vector genome encoded by plasmid Ps-AAV-ITR / minCBA-HI-cLub1co-BGH. 45. The method described in any of items 19-44, wherein rAAV is administered intraarticularly. 46. ​​A recombinant plasmid vector containing a nucleic acid sequence encoding a canine HAS2 polypeptide ligated to a promoter. 47. A recombinant plasmid according to item 46, wherein the nucleic acid sequence encoding the HAS2 polypeptide is at least 90% identical to the sequence shown in SEQ ID NO: 3, or the nucleic acid encodes a HAS2 polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2. 48. A recombinant plasmid as described in item 46 or 47, wherein the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. 49. A recombinant plasmid containing pCBA-HI-cHAS2-BGHpA, as described in any of items 46-49. 50. A recombinant plasmid vector containing a nucleic acid sequence encoding a truncated inulbricin, which is ligated to a promoter. 51. A recombinant plasmid according to item 50, wherein the nucleic acid sequence encoding rubrisin is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 6, or the nucleic acid encodes rubrisin containing an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7. 52. A recombinant plasmid according to item 50 or 51, wherein the rubrisin polypeptide has the amino acid sequence shown in SEQ ID NO: 7. 53. A recombinant plasmid described in either item 46-49 or 50-52, wherein the promoter is selected from the group consisting of the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter. 54. A recombinant AAV5 virus vector containing the nucleotide sequence shown in Sequence ID No. 8. 55. rAAV, including the rAAV vector described in item 53. 56. A pharmaceutical composition comprising the rAAV described in item 55, and at least one pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle. 57. A method for treating a target mammal suffering from osteoarthritis, comprising the step of intra-articular administration of a therapeutically effective amount of the pharmaceutical composition described in item 56 to the target mammal. 58. The method described in 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 joint osteoarthritis (OA) in mammals by long-term gene expression of HAS2 or rubrisin in synovial cells and / or chondrocytes, wherein the system comprises an rAAV vector comprising a nucleic acid sequence encoding HAS2 or rubrisin, and left and right AAV reverse terminal repeats (L ITR and R ITR), wherein the expression of the HAS2 or rubrisin gene in synovial cells and / or chondrocytes is regulated by a promoter, the promoter is located upstream of the reading frame of the nucleic acid sequence encoding HAS2 or rubrisin, and the promoter is further specifically activated by an increase in the level of an immunostimulant. 60. The AAV system described in item 59, where HAS2 is a mammalian HAS2. 61. An AAV system as described in item 59 or 60, in which HAS2 is human HAS2. 62. An AAV system described in any of items 59-61, whose promoter is an inducible inflammation promoter. 63. The AAV system described in item 62, wherein the inducible inflammation promoter is selected from the 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 a hybrid construct thereof. 64. An AAV system as described in any of items 59-63, wherein the rAAV vector genome comprises HAS2 containing the amino acid sequence of SEQ ID NO: 2, rubrisin containing the amino acid sequence of SEQ ID NO: 7, or a nucleic acid encoding the functional variant of the above. 65. An AAV system described in any of items 59-64, wherein the rAAV vector genome includes a marker gene that enables monitoring of the vector genome in synovial cells and / or chondrocytes. 66. An AAV system as described in any of items 59-65, wherein the rAAV vector genome contains a nucleic acid having at least 80% or 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6. 67. An AAV system described in any of items 59-66, wherein the rAAV vector genome contains the nucleic acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6. 68. An AAV system described in any of items 59-67 for the treatment or prevention of osteoarthritis. 69. A pharmaceutical composition containing an AAV system as described in any of items 59-68. 70. An rAAV comprising an rAAV vector, the rAAV comprising a nucleic acid sequence encoding a canine HAS2 polypeptide ligated to a promoter. 71. The rAAV according to item 70, wherein the nucleic acid sequence encoding the HAS2 polypeptide is at least 90% identical to the sequence shown in SEQ ID NO: 3, or the nucleic acid encodes a HAS2 polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2. 72. The rAAV described in item 70 or 71, wherein the HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. 73. An rAAV vector containing a nucleic acid sequence encoding inulbricin, which is ligated to a promoter so as to be activated. 74. The rAAV according to item 73, wherein the nucleic acid sequence encoding rubrisin is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 6, or the nucleic acid encodes rubrisin having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 7. 75. The rAAV described in item 73 or 74, wherein the rubrisin polypeptide has the amino acid sequence shown in SEQ ID NO: 7. 76. An AAV vector containing the nucleotide sequence shown in Sequence ID: 8, as described in any of items 50-53. 77. rAAV as described in either item 71-72 or 74-76, wherein the promoter is selected from the group consisting of the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter. 78. rAAV as described in any of items 71-77, including an AAV2 capsid or an AAV5 capsid. 79. A pharmaceutical composition comprising an rAAV as described in any of items 71-78, and at least one pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle. 80. A method for treating a target mammal suffering from osteoarthritis, comprising the step of intra-articular administration of a therapeutically effective amount of the pharmaceutical composition described in item 79 to the target mammal. 81. The method described in item 80, wherein the target mammal is human or dog. 82. An isolated nucleic acid having the sequence shown in Sequence ID: 4. 83. An isolated polypeptide having the sequence shown in Sequence ID No. 5. The present invention will now be expressed in accordance with the following non-limiting claims.

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising an rAAV comprising a nucleic acid sequence encoding a canine hyaluronic acid synthase 2 (HAS2) polypeptide ligated to a promoter, and comprising an AAV5 capsid.

2. The rAAV according to claim 1, wherein the canine HAS2 polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

2.

3. The rAAV according to claim 1 or 2, wherein the canine HAS2 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

2.

4. The rAAV according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding the canine HAS2 polypeptide is codon-optimized.

5. The rAAV according to any one of claims 1 to 4, wherein the nucleic acid sequence encoding the canine HAS2 polypeptide is at least 90% identical to the sequence shown in SEQ ID NO:

3.

6. rAAV according to any one of claims 1 to 5, wherein the promoter is selected from the group consisting of the CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothionein gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter.

7. The rAAV according to any one of claims 1 to 6, wherein the rAAV comprises an rAAV vector genome having the following elements from 5' to 3': 5'AAV reverse terminal repeat (ITR), stuffer nucleic acid, promoter, intron (IN), nucleic acid sequence encoding a canine HAS2 polypeptide, polyadenylation signal (pA), and 3'AAV ITR.

8. The rAAV according to any one of claims 1 to 7, comprising an expression cassette containing the following elements from 5' to 3': a chicken β-actin promoter, a hybrid intron, a nucleic acid sequence encoding a canine HAS2 polypeptide, and a bovine growth hormone polyA moiety.

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