Use of fibrin hydrogels for localized and cell-controlled lentiviral gene delivery.

摘要

Recombinant lentiviruses are efficient gene delivery vehicles with the ability to infect non-dividing cells including neurons, hematopoietic and embryonic stem cells. In addition to high efficiency several applications may require localized and cell-controlled gene transfer to a subset of cells in a local microenvironment. To this end, we developed a strategy of lentivirus delivery using fibrin hydrogels. Our preliminary studies indicate that fibrin-mediated gene transfer is more efficient than traditional lentivirus transduction and that transduction efficiency depends on the properties of fibrin gels and the interaction of target cells with the matrix. Although transduction efficiency is highest at low fibrinogen concentrations, the lentiviral particles diffuse out, decreasing transduction efficiency. We hence conclude that lentiviral transduction is optimum at intermediate concentrations of fibrinogen but interestingly it is independent of thrombin. Interestingly, fibrinolytic inhibitors decreased lentivirus transduction in a dose dependent manner, suggesting that matrix degradation is necessary for virus uptake. We optimized the protocol to incorporate fibronectin and polybrene in the gels for wider applicability. Using this strategy we engineered microarrays with immobilized lentivirus to express multiple genes in a single transduction step. This high throughput gene delivery strategy will have significant impact on many biological studies including profiling of adult and embryonic stem cells.