Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis

Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200–800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA. Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200–800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA.