gms | German Medical Science

Objectives: rAAV vectors are attractive tools for the durable treatment of human osteoarthritis (OA). Still, their adapted use in vivo is impaired by neutralizing antibodies against viral capsid elements in patients. Delivery of rAAV via polymeric micelles may allow overcoming these hurdles. Here, we tested the feasibility of targeting human OA chondrocytes via rAAV using poly(ethylene oxide) (PEO) and poly(propylene oxide) (PEO-PPO-PEO) polymeric micelles in vitro and in a model of osteochondral defect in situ.

Methods: rAAV-lacZ carries the E. coli β -galactosidase gene (lacZ) controlled by the CMV-IE promoter/enhancer. OA cartilage was obtained from joints of patients undergoing total knee arthroplasty. Cells (hOACs) were isolated from unprocessed OA cartilage. Experimental human osteochondral defects (OCD) were created using a 1-mm drill needle in standardized cylindrical osteochondral explants. PF68 or T908 (BASF) polymeric micelles were mixed with rAAV-lacZ and directly applied to the cells or defects. Control conditions included similar amounts of rAAV in sucrose or sucrose 10%. Delivery of rAAV from polymeric micelles was evaluated using the AAV Titration ELISA. Virus-micelle binding studies were performed by isothermal titration calorimetry (ITC). Alternatively, rAAV/PF68 and rAAV/T908 micelles were incubated with an anti-AAV A20 capsid antibody prior to testing. Transgene expression was monitored by X-Gal staining and by Beta-Glo® assay. Each condition was performed in duplicate in two independent experiments. The t-test and ANOVA were employed to assess statistical differences (P ≤ 0.050).

Results and Conclusion: Diffusion of free vector led only to the detection of ~ 75% of the total amount of rAAV loaded after 10 days versus 100% when rAAV was delivered via polymeric micelles. Higher levels of transgene expression were noted when rAAV-lacZ was formulated in the micelles compared with free vector treatment (45- and 78-fold difference for PF68 and T908, respectively, P ≤ 0.040). Interaction of rAAV with the micelles was confirmed by ITC. Addition of the A20 antibody significantly impaired gene transfer via rAAV in cells relative to conditions where A20 was omitted (up to 2-fold, P ≤ 0.050). Delivery of rAAV via PF68 or T908 micelles in the presence of A20 restored gene transfer to levels similar to those achieved upon free vector treatment without A20 (up to 2.3-fold, P ≤ 0.040). Both PF68 and T908 micelles promoted higher intensities of X-Gal staining in lacZ-treated OCDs than in samples treated with free rAAV-lacZ in the presence of A20 (P = 0.001). Delivery of rAAV via PEO-PPO-PEO polymeric micelles thus allows to increase both the stability and bioactivity of rAAV, promoting high levels of transgene expression while affording protection against antibody neutralization