PEO–PPO–PEO micelles as effective rAAV-mediated gene delivery systems to target human mesenchymal stem cells without altering their differentiation potency

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

“…The obtained poloxamer and poloxamine solutions were then mixed with rAAV (or Cy3-labeled rAAV; 10 10 transgene copies/mL) in equal volumes, kept in ice-water bath for 30 min, and used. 36 The final copolymer concentration into the medium was always 2%. 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). Application of rAAV carrying a reporter (lacZ) gene was not included in this study due to its absence of effects on chondrocytes metabolic activities in accordance with our previous studies.…”

“…36,40 Controls included cells maintained with or without same dose of free vector, and also with copolymer in the absence of rAAV.…”

“…34,35 In this regard, we previously reported that polymeric micelles that rely on poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) copolymers in the form of linear poloxamers or X-shaped poloxamines may improve the stability and bioactivity of reporter (lacZ) rAAV gene vectors, especially those with a higher PEO/PPO ratio (poloxamer PF68 and poloxamine T908). 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.…”

“…32,36,40 The vectors were packaged as conventional (not self-complementary) vectors using a helper-free, 2-plasmid transfection system in 293 cells with the packaging plasmid pXX2 and the adenovirus helper plasmid pXX6. 32 The vector preparations were purified by extensive dialysis and titrated by real-time polymerase chain reaction, 32,36,40 averaging 10 10 transgene copies/mL (~1/500 functional recombinant viral particles).…”