Feasibility of intra-articular adeno-associated virus-mediated proteoglycan-4 gene therapy to prevent osteoarthritis

<p>Lubricin, or proteoglycan 4 (PRG4), is a secreted, glycosylated protein that binds to cartilage surfaces, which functions as a boundary lubricant. The loss of lubricin from cartilage is identified as a major pathogenic factor in post-traumatic osteoarthritis (PTOA) that has now been the aim of therapeutic intervention. Intra-articular injection of PRG4 protein provides short-term benefits that might be extended using sustained delivery methods such as in gene therapy.</p> <p>Here we describe the development and testing of such therapy using adeno-associated virus (AAV) as a vector for the transfer of PRG4-green fluorescent protein (GFP) fusion gene. Our recombinant PRG4 gene produces a PRG4-GFP fusion protein to facilitate tracking of its expression and distribution on joint surfaces. We hypothesized that PRG4-GFP is fully functional as a cartilage lubricant and that PRG4-GFP produced <em>in vivo</em> is expressed by synoviocytes and other joint cells, and cartilage surfaces remained coated for several weeks up to months after intra-articular injection of the virus.</p> <p>PRG4-GFP showed lubricin-like cartilage binding <em>in vitro</em>, and lubrication immunoblot analysis confirmed that purified PRG4-GFP from cultured media conditioned by PRG4-GFP-transduced synoviocytes was heavily glycosylated, while confocal microscopy revealed binding of the fluorescent fusion protein to cartilage surfaces. Metal-on-cartilage friction tests showed that PRG4-GFP reduced friction coefficients to a degree comparable to that of synovial fluid and had strong chondro-protective effects in explanted cartilage exposed to shear loading. The chondrocyte viability after shear loading showed that PRG4-GFP had a strong chondro-protective effect on par with that of the synovial fluid. Confocal microscopy and immunohistology confirmed that cartilage surfaces in the stifle joints of mice injected with viruses were coated with PRG4-GFP for up to 2 or 4 weeks after the treatment. The overexpression of PRG4-GFP and coating of cartilage surfaces in the stifle joints of mice injected with Adeno-Associated Virus for the transfer of PRG4-GFP fusion gene (AAV-PRG4-GFP) was confirmed by confocal microscopy and immunohistology for up to 2 or 4 weeks post-injection. The μCT imaging and immunohistology in AAV-PRG4-GFP injected rabbit knees showed stronger inhibition in degeneration of damaged tissues than in AAV-GFP injected control group. Collectively these findings indicate that AAV-PRG4-GFP transduction is a valuable new tool for evaluating the effects of long-term lubricant supplementation on PTOA in animal models.</p>