gms | German Medical Science

Objectives: Articular cartilage has a poor capacity for self healing. Gene therapy allows for the application of therapeutic genes in sites of cartilage injury, resulting in a temporarily and spatially defined delivery of the candidate agent. rAAV vectors are particularly clinically adapted tools to achieve this goal. Important limitations to their use still include a relatively low efficacy of cell penetration (~ 13%), the presence of neutralizing antibodies against the viral capsid, and presence of inhibitory anticoagulants in samples provided from the clinics. Delivery of rAAV via nonclassical routes of transduction is thus under active investigation. Here, we evaluated the effects of delivering rAAV vectors to human bone marrow-derived mesenchymal stem cells (hMSCs) in the presence of linear via poly-ethylene oxide (PEO) and poly-propylene oxide block copolymers (PPO) triblock copolymers (Pluronic®) and X-shaped counterparts (Tetronic®).

Methods: rAAV-lacZ carries the E. coli β-galactosidase gene (lacZ) and rAAV-RFP the Discosoma sp. red fluorescent protein gene (RFP). Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty for hMSC isolation. Poloxamers (Pluronic®) PF68 and PF127 or Poloxamines (Tetronic®) 908, 1107, 1301, and 1307 were mixed with rAAV and added to the cultures while free rAAV solutions were used as controls. Transgene expression was monitored by detection of live fluorescence, X-Gal staining, and with the Beta-Glo® Assay System. Cell viability was monitored with the Cell Proliferation reagent WST-1. The effects of the copolymers upon hMSC transduction were also tested in the presence of heparin that prevents vector uptake. Each condition was performed in duplicate in two independent experiments. The t-test was employed with P below 0.05 considered statistically significant.

Results and Conclusion: Efficient, stable RFP expression was noted over time in rAAV-RFP-transduced hMSCs upon contact with the different copolymers, with the strongest signal obtained for T908 and T1107, even higher than when adding rAAV-RFP as a free vector solution. Effective, prolonged gene transfer was also evidenced in rAAV-lacZ-transduced hMSCs by Beta-Glo® assay, with superior efficacy of T908, T1107, and T1307. Cell viability after 21 days of culture ranged between 35-100%, with PF68 and T908 as the most tolerated treatments. Strikingly, while application of free vector solutions was strongly inhibited in the presence of heparin, co-administration of heparinized vectors with the different copolymers increased the cellular uptake and bioactivity of such modified vectors, except for the most hydrophobic T1301 copolymer. PEO-PPO copolymers are thus highly promising materials for rAAV delivery, allowing to achieve high levels of transgene expression. Evaluations are ongoing to examine the mechanisms of action of these compounds.