PEO-PPO-PEO Carriers for rAAV-Mediated Transduction of Human Articular Chondrocytes in Vitro and in a Human Osteochondral Defect Model

Abstract

Gene therapy is an attractive strategy for the durable treatment of human osteoarthritis (OA), a gradual, irreversible joint disease. Gene carriers based on the small human adeno-associated virus (AAV) exhibit major efficacy in modifying damaged human articular cartilage in situ over extended periods of time. Yet, clinical application of recombinant AAV (rAAV) vectors remains complicated by the presence of neutralizing antibodies against viral capsid elements in a majority of patients. The goal of this study was to evaluate the feasibility of delivering rAAV vectors to human OA chondrocytes in vitro and in an experimental model of osteochondral defect via polymeric micelles to protect gene transfer from experimental neutralization. Interaction of rAAV with micelles of linear (poloxamer PF68) or X-shaped (poloxamine T908) poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers (PEO-PPO-PEO micelles) was characterized by means of isothermal titration calorimetry. Micelle encapsulation allowed an increase in both the stability and bioactivity of rAAV vectors and promoted higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defects compared with that of free vector treatment without detrimental effects on the biological activity of the cells or their phenotype. Remarkably, protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPO-PEO micelles in all systems evaluated, especially when using T908. Altogether, these findings show the potential of PEO-PPO-PEO micelles as effective tools to improve current gene-based treatments for human OA.

rAAV-mediated overexpression of TGF-β via vector delivery in polymeric micelles stimulates the biological and reparative activities of human articular chondrocytes in vitro and in a human osteochondral defect model

“…Cells were incubated at the denoted cell densities in DMEM, 10% fetal bovine serum, 100 U/mL penicillin G, 100 µL/mL streptomycin (growth medium) for 12 h at 37°C under 5% CO 2 prior to addition of the rAAV/copolymer systems or free rAAV preparations (see below for concentrations) for up to 10 days for consistency with our previous study with reporter vectors. 37 Osteochondral defects were created in human OA cartilage biopsies (n=7) using a 1-mm drill needle in standardized cylindrical (6-mm diameter) as previously described 37 and incubated in growth medium prior to addition of the rAAV/ copolymer systems or free rAAV preparations at the concentrations indicated thereafter for 10 days.…”

“…Characterization of the systems revealed an effective association between the vectors and the polymeric micelles, also supported by the increase in size of the aggregates as recorded by transmission electron microscopy and dynamic light scattering. 37 Gene transfer efficacy using the rAAV/ copolymer systems Monolayer cultures of human OA chondrocytes (3,000 cells/ well in 96-well plates) and osteochondral defect cultures were directly incubated with the rAAV/PF68 or rAAV/T908 micelles (20 or 40 µL for monolayer cultures; 100 µL for osteochondral defect cultures; final copolymer concentration 2%) and kept for 10 days at 37°C with 3 weekly medium change. 36,40 Control conditions included application of 10% sucrose aq solution (negative control), similar amounts of free rAAV (10 or 20 µL for monolayer cultures; 40 µL for osteochondral defect cultures; 10 10 transgene copies/mL) in 10% sucrose (v/v; positive control), and copolymer with 10% sucrose (w/v; copolymer control).…”

“…37 Fixed cells were stained with Alcian blue (matrix proteoglycans) and excess stain was washed off with double distilled water. The stain was quantified by overnight solubilization in 6 M guanidine hydrochloride to monitor absorbance at 595 nm (GENios, Tecan).…”

“…36 In addition, encapsulation of rAAV in PF68 or T908 polymeric micelles resulted in an effective gene transfer of the reporter gene lacZ in human OA chondrocytes in vitro and in experimental osteochondral defects without detrimental effects on the biological activities of the cells nor on their phenotype, also affording protection when anti-AAV capsid neutralizing antibodies were present. 37 In light of such promising findings, the purpose of the present study was to test whether PF68 and T908 polymeric micelles can deliver a candidate rAAV TGF-β vector to human OA chondrocytes and to human osteochondral defects in order to overexpress the growth factor as a potent therapeutic approach for the future treatment of articular cartilage injuries.…”

Gene therapy for chondral and osteochondral regeneration: is the future now?

“…Various joint pathologies such as rheumatic disease, trauma, osteochondritis dissecans and osteonecrosis may lead to severe damage of articular cartilage and other joint structures, ranging from focal defects to osteoarthritis (OA) [1]. The critical actor in this pathophysiological process is the osteochondral unit.…”