gms | German Medical Science

Objectives: rAAV are clinically adapted gene vectors for human regenerative medicine yet their use is still associated with a moderate cellular uptake due to host humoral immune responses and in the presence of inhibitory clinical compounds like heparin. Here, we tested the possibility of delivering rAAV to human bone marrow-derived mesenchymal stem cells (hMSCs) via polymeric micelles made of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) triblock copolymers as linear poloxamers (Pluronic®) (PF68) or as X-shaped poloxamines (Tetronic®) (T908).

Methods: rAAV-lacZ carries the E. coli β -galactosidase gene (lacZ) and rAAV-FLAG-hsox9 a FLAG-tagged human sox9 cDNA. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty for hMSC isolation. Cells were incubated in growth medium or pelleted to form aggregate cultures in chondrogenic medium. Poloxamer PF68 and poloxamine T908 were prepared and mixed with rAAV prior to addition to the cultures. Free rAAV preparations were used as controls. hMSCs were also incubated with rAAV/PF68 or rAAV/T908 in the presence of heparin (2.5 I.U.) or with an AAV-specific antibody (A20, 1:500). Transgene expression was monitored by X-Gal staining and using the Beta-Glo® Assay System. Chondrogenesis was assessed by histological, immunohistochemical, and real-time RT-PCR analyses. Each condition was performed in duplicate in three independent experiments. The t-test was employed with P ≤ 0.05 considered statistically significant.

Results and Conclusion: PF68 and T908 micelles allowed for the successful modification of hMSCs in monolayer culture using rAAV-lacZ relative to free vector treatment (up to 2.7-fold difference, P ≤ 0.030). Delivery of heparinized rAAV via T908 restored lacZ transduction compared with free heparinized vector treatment (up to 4.2-fold difference, P ≤ 0.040). While gene transfer was reduced in the presence of the A20 AAV-specific antibody (55% versus 100% without A20 upon free vector treatment, a 1.8-fold decrease, P = 0.020), administration of rAAV via PF68 and T908 restored gene transfer in the presence of A20 relative to free vector treatment (100% and 95%, P ≥ 0.250 and P ≤ 0.001, respectively). Chondrogenic differentiation was noted in all samples, especially when delivering a sox9 vector versus lacZ. Application of rAAV sox9 via PF68 and T908 enhanced SOX9 (~ 8.5-fold difference, P ≤ 0.020), ACAN (4.3-fold difference, P = 0.029), and COL2A1 expression (1.1-fold difference, P = 0.425) relative to free vector treatment. These findings highlight the value of PEO-PPO-PEO micelles as powerful tools for rAAV-based regenerative medicine.