Evaluation of PRG4 mRNA-loaded polymeric nanoparticles in vitro and in vivo models of osteoarthritis

BACKGROUND: Osteoarthritis (OA) is a highly prevalent degenerative joint disease for which no disease-modifying treatments are available. Proteoglycan 4 (PRG4) is a glycoprotein with lubricating and immunomodulatory properties, making it a promising therapeutic target for mRNA-based therapies. Therefore, this study evaluated the effects of PRG4 mRNA delivered via polymeric nanoparticles in both in vitro and in vivo OA models. METHODS: Human chondrocytes from OA patients were cultured in 3D pellet models and transfected with PRG4 mRNA-loaded polymeric nanoparticles at low or high dose, once or twice, respectively. Viability, pellet growth, glycosaminoglycan (GAG) production and mRNA expression were assessed, with EGFP mRNA transfection as a negative control. In vivo, an osteoarthritis rat model was used to evaluate PRG4 mRNA effects on pain behavior, cartilage degeneration, and synovial inflammation. RESULTS: In vitro, nanoparticle treatment induced an increase in pellet size and overall GAG production, independent of their cargo. PRG4 mRNA nanoparticle treatment at lower doses increased total pellet GAG content and early GAG release. Higher doses showed no additional benefit, and repeated transfection of the high dose significantly decreased cell viability. Double transfection of either dose showed increased relative expression of PRG4 mRNA, indicating uptake of the nanoparticles into the pellet. In vivo, PRG4 mRNA nanoparticles did not alleviate pain, reduce OA severity, or mitigate synovial inflammation. Sham-operated joints injected with mRNA-loaded nanoparticles showed increased OA and inflammation scores compared to healthy joints. CONCLUSIONS: MRNA-loaded nanoparticles increased extracellular matrix production in vitro independent of mRNA sequence. A minor effect of delivering PRG4 mRNA compared to control mRNA was found in matrix production normalized to cell content, but this was not reproduced in vivo. An intrinsic effect of nanoparticles on matrix production is suggested. Further research into the biodistribution of the presented nanoparticles and their cellular uptake into the different joint tissues is necessary for the advancement of mRNA-based treatments of OA.