Recombinant AAV Vectors Expressing Osteoprotective genes, including HAS2 and Lubricin, useful in the Treatment of Osteoarthritis and Related Joint Conditions in Mammals

Abstract

The present disclosure relates to recombinant viral vectors, to pharmaceutical compositions comprising such recombinant vectors, and to methods for prevention and treatment of osteoarthritis in mammals. In particular, 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 AAV 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.

What is claimed: 
     
         1 . A recombinant adeno-associated virus (rAAV) comprising a rAAV vector, wherein the rAAV vector comprises a nucleic acid sequence encoding a canine HAS2 polypeptide operably linked to a promoter. 
     
     
         2 . The rAAV of  claim 1 , wherein the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity to the sequence as set forth in SEQ ID NO:3 or the nucleic acid encodes a HAS2 polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, or wherein the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2; and/or the recombinant plasmid comprises pCBA-HI-cHAS2-BGHpA. 
     
     
         3 . A rAAV comprising a rAAV vector, wherein the rAAV vector comprises a nucleic acid sequence encoding a shortened canine lubricin operably linked to a promoter. 
     
     
         4 . The rAAV of  claim 3 , wherein the nucleic acid sequence encoding the lubricin has at least 90% identity to the nucleotide sequence set forth in SEQ ID NO: 6 or the nucleic acid encodes a lubricin comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. 
     
     
         5 . The rAAV of  claim 4 , wherein the lubricin polypeptide has an amino acid sequence as set forth in SEQ ID NO: 7. 
     
     
         6 . The rAAV of  claim 4 , wherein the rAAV vector comprises the nucleotide sequence as set forth in SEQ ID NO: 8. 
     
     
         7 . The rAAV of  claim 2 , wherein the promoter is selected from the group consisting of a CMV IE promoter, a RSV promoter, an HSV-1 TK promoter, a SV40 early promoter, a SV40 late promoter, a phosphoglycerate kinase gene promoter, a metallothionein gene promoter, an α-1 antitrypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a CBA promoter, a β-actin gene promoter, a β-globin gene promoter, a γ-globin gene promoter, an α-fetoprotein gene promoter, and a muscle creatine kinase gene promoter. 
     
     
         8 . The rAAV of  claim 2 , wherein the rAAV comprises an AAV2 capsid or a AAV5 capsid. 
     
     
         9 . The rAAV of  claim 8 , wherein the rAAV comprises an AAV5 capsid. 
     
     
         10 . A pharmaceutical composition comprising the rAAV of  claim 1 , and optionally at least one pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle. 
     
     
         11 . A method of treating a mammalian subject suffering from osteoarthritis (OA), comprising intra-articularly administering to said mammalian subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding an osteo-protective or osteo-regenerative polypeptide operably linked to a promoter, wherein the polypeptide is expressed in vivo in the mammalian subject in an amount effective to alleviate the symptoms of OA;
 wherein the polypeptide is a hyaluronic acid synthase (HAS), i a lubricin, an Interleukin-1 Receptor (IL-1R) antagonist, an Insulin-like growth factor 1 (IGF-1), a fibroblast growth factor 2 (FGF-2), a Transforming growth factor beta 1 (TGFβ1), a Bone Morphogenetic protein 7 (BMP7), a Glucosamine-fructose-6-phosphate aminotransferase (GFAT), an Interleukin 10 (IL-10), a heme oxygenase-1 HO-1, biologically active truncations thereof, or combinations thereof.   
     
     
         12 . The method of  claim 11 , wherein the polypeptide is a HAS2 polypeptide. 
     
     
         13 . The method of  claim 11 , wherein the HAS2 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 2, or a fragment, a variant, or a homolog thereof which exhibits HAS2 activity in vivo in the subject. 
     
     
         14 . The method of  claim 13 , wherein the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. 
     
     
         15 . The method of claim ii, wherein the nucleic acid encoding the HAS2 polypeptide has a nucleotide sequence having at least 9o% identity to the nucleotide sequence set forth in SEQ ID NO: 3; and
 wherein the rAAV comprises a rAAV vector genome comprising from 5′ to 3′ the following elements: 5′ AAV inverted terminal repeat (ITR), stuffer nucleic acid, a promoter, an intron (IN), a cHAS2 codon-optimized cDNA, a polyadenylation signal (pA), and a 3′ AAV ITR.   
     
     
         16 . A method of increasing the production of hyaluronic acid in chondrocytes and/or synoviocytes of a canine, comprising the steps of administering a rAAV to the canine, wherein the rAAV comprises a rAAV vector genome comprising nucleic acid encoding an HAS2 enzyme operably linked to a promoter, and wherein following administration the HAS2 enzyme is expressed and catalyzes the production of additional hyaluronic acid, thereby increasing the level of hyaluronic acid (HA) in the canine. 
     
     
         17 . The method of  claim 16 , wherein the HAS2 is produced in sufficient quantity to treat the symptoms of OA in canine. 
     
     
         18 . The method of  claim 16 , wherein the HA levels are restored to levels found in healthy canines. 
     
     
         19 . The method of  claim 16 , wherein the nucleic acid encoding the HAS2 polypeptide has a nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in SEQ ID NO: 3; and
 wherein the rAAV comprises a rAAV vector genome comprising from 5′ to 3′ the following elements: 5′ AAV inverted terminal repeat (ITR), stuffer nucleic acid, a promoter, an intron (IN), a cHAS2 codon-optimized cDNA, a polyadenylation signal (pA), and a 3′ AAV ITR.   
     
     
         20 . The method of  claim 19 , wherein the nucleic acid encoding the HAS2 polypeptide has a nucleotide sequence as set forth in SEQ ID NO: 3; and
 wherein the rAAV comprises a rAAV vector genome comprising from 5′ to 3′ the following elements: 5′ AAV inverted terminal repeat (ITR), stuffer nucleic acid, a promoter, an intron (IN), a cHAS2 codon-optimized cDNA, a polyadenylation signal (pA), and a 3′ AAV ITR.