Thermosensitive hydrogel for prolonged delivery of lentiviral vector expressing neurotrophin-3 in vitro.

BACKGROUND The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/β-glycerol phosphate (β-GP) as a carrier. METHODS Three chitosan/β-GP scaffolds were prepared with varying concentrations of chitosan and β-GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin-3 (Lenti NT-3) was incorporated into the chitosan/β-GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of pre-seeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence-activated cell sorting analysis. The effect of Lenti NT-3 was analyzed by measuring DRG neurite outgrowth. RESULTS Collagen displayed its most significant elution of virus on day 1 and chitosan/β-GP (with a final concentration of 2.17% chitosan) on day 3. CONCLUSIONS The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long-term gene expression for therapeutic factors to treat conditions such as injury to the nervous system.