Gene delivery into primary brain capillary endothelial cells for protein secretion:a novel strategy for drug delivery to the brain

Central nervous system (CNS) diseases are becoming more prevalent. Unfortunately, the treatment of CNS diseases is often rendered complicated by the inability of many drugs of therapeutic relevance to cross the blood-brain barrier (BBB). The BBB is formed by non-fenestrated brain capillary endothelial cells (BCECs) and their intermingling tight junctions. In order to enhance drug delivery to the brain, different approaches have been developed. A promising and novel approach to overcome the restricting properties of the BBB is gene therapy. Gene therapy is based on the delivery of genetic material into BCECs, which, theoretically, will result in expression and secretion of the recombinant protein from the BCECs and into the brain, thus turning BCECs into small recombinant protein factories. The aim of this study was to investigate the possibility of using BCECs as small factories for recombinant protein production. We have previously shown that it is possible to transfect human microvascular endothelial cells (HBMEC) and rat brain endothelial (RBE4) cells with genetic material encoding the human growth hormone (GH1) (Thomsen et al 2011). In the present study, however, an in-vitro BBB model was established by co-culturing primary BCECs together with primary astrocytes, both of which were isolated from rats. This was done in order to study the possibility of using gene transfection in an environment closer to the in-vivo BBB situation.The in-vitro BBB barrier model showed trans-endothelial electrical resistance above 200 ohm*cm2, indicating that the BCECs formed a tight polar monolayer with functional tight junctions. This was confirmed by immunostaining for the thigh junction protein ZO-1. Rat BCECs were transfected with a red fluorescence protein Hc-RED for 24 hours. Positive transfection and protein expression within the BCECs were detected by fluorescence microscopy. The Barrier integrity was assessed during the 24h transfection period.