Viral gene therapeutic strategies to obtain abluminal protein secretion from brain endothelial cells denoting the blood-brain barrier

The blood-brain barrier (BBB) formed by brain endothelial cells (BECs) limits the passage of biopharmaceuticals from the circulation into the brain parenchyma. An emerging strategy suggests that viral vectors may specifically target and productively transduce BECs leading to protein secretion into the brain. To study protein secretion, different AAV-BR1 vector constructs encoding mCherry were designed containing either an endothelial specific promotor or the more ubiquitous CAG promotor. Intending to enhance abluminal secretion, a fragment of platelet-derived growth factor subunit B (PDGF-B) protein was fused to the N-terminus of mCherry. Post-translational removal of PDGF-B using a furin cleavage site was also examined. Secretion of mCherry from BECs occurred primarily without post-translational processing, hence limiting the applicability of PDGF-B fusion to proteins among which bioactivity is not abolished by N-terminal modifications. Modelling the BBB in vitro using primary BECs co-cultured with glial cells, mCherry secretion across the abluminal membrane was particularly favorable, following transduction with constructs containing the CAG promoter and PDGF-B fragment. Systemically administered AAV-BR1 vector transduced BECs in vivo, but tracing mCherry secretion into the brain was complicated by additional BBB crossing of the vector with neuronal off-target transduction. In conclusion, the AAV-BR1 vectors transduce BECs and enable abluminal protein secretion with potential for protein delivery to the brain.