Benefits of photopolymerizable hydrogels for the effective delivery of raav vectors in human bone marrow derived mesenchymal stem cells

Abstract

Purpose: Injured adult articular cartilage has a limited ability for self-healing. Administration of genetically modified bone marrow-derived mesenchymal stromal cells (MSCs) in cartilage defects is a promising approach to enhance cartilage repair, especially when using clinically relevant recombinant adeno-associated virus (rAAV) vectors [1,2]. Yet, the presence of neutralizing antibodies against the viral capsid proteins in patients [3] may impair effective gene transfer in translational protocols. Scaffold-guided rAAV gene transfer has the potential to overcome such an issue by protecting the vectors from neutralization while increasing their gene transfer efficacy by controlling their spatial and temporal delivery [4-7]. Here, we examined the feasibility of delivering rAAV vectors in human MSCs (hMSCs) via photopolymerizable hydrogel systems [8,9] as future, effective platform for cartilage repair. Methods: rAAV vectors were packaged, purified, and titrated as previously described [10,11]. rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene and rAAV-RFP the Discosoma sp. red fluorescent protein gene (RFP) [10,11]. Cy3 labeling of the rAAV vectors was performed using a Cy3 Ab Labeling Kit (Amersham/GE Healthcare) with detection by live fluorescence imaging as previously described [12]. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty (n = 2). hMSCs were prepared as previously described [10,11]. Cells (passage 1) were seeded in 48-well plates (5,000 cells/well), maintained in DMEM, 10% FBS, 100 U/ml penicillin G, 100 μl/ml streptomycin, and incubated at 37ºC during 12 h before addition of the various hydrogel systems. The hydrogel systems (FID119 F3 and FID119 F4) were prepared by overnight dissolution of the compounds (300 mg of FID119 F3, i.e. F3; 200 mg of FID119 F4, i.e. F4) 10 ml NaCl 0.9% (w/v). The F3 and F4 hydrogel systems were mixed with rAAV-lacZ or rAAV-RFP in equal parts using a BTC® Medical Europe UV-Lamp (5 min) and directly applied to the cultures for up to 21 days. Release of rAAV from the hydrogels was evaluated by AAV Titration ELISA (Progen) in conditioned culture medium using a permeable support system as previously described [12]. Transgene (RFP) expression was detected by live fluorescence imaging [10,11]. Cell viability was monitored using the Cell Proliferation reagent WST-1 (Roche Applied Science) [12]. Each condition was performed in duplicate in two independent experiments. Results: The F3 and F4 hydrogels (Fig. 1A) were both capable of encapsulating rAAV as noted by effective detection of Cy3 vector labeling in the hydrogel systems relative to control conditions (encapsulation of unlabeled vectors) (Fig. 1B). An analysis of the rAAV release profiles (Fig. 1C) showed that both the F3 and F4 hydrogels effectively released rAAV over a prolonged period of time (21 days, the longest time-point evaluated) compared with hydrogels lacking rAAV vectors, with hydrogel-free rAAV vectors, and with a condition lacking both the hydrogel and rAAV. Specifically, the F4 hydrogel system was the most potent to support a controlled release of rAAV over time (Fig. 1C). An estimation of transgene (RFP) expression upon direct contact of the hydrogels with hMSCs over time revealed that rAAV-RFP released from the F3 and F4 hydrogels promoted an effective overexpression of the RFP reporter gene in the cells for at least 21 days (the longest time-point examined) versus the various control conditions tested (Fig. 2). Equally important, an analysis of the cell proliferation indices in the cells showed no deleterious effects of the F3 or F4 hydrogel systems carrying rAAV relative to the various control conditions tested (Fig. 3). Conclusions: Controlled delivery of rAAV via FID119 hydrogels (especially with the F4 hydrogel system) has the potential to safely and durably modify hMSCs. Combining rAAV gene transfer with the use of photopolymerizable (FID119) hydrogels may provide novel, promising tools to enhance cartilage repair by implanting MSCs modified to overexpress therapeutic (chondroregenerative) genes via controlled delivery of rAAV using in particular the F4 hydrogel. 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