A novel vector design targeting the blood-brain barrier with the brain-specific AAV-BR1 vector enables abluminal protein secretion from brain endothelial cells in vitro.

BACKGROUND: The blood-brain barrier (BBB) formed by brain endothelial cells (BECs) limits the passage of biopharmaceuticals from the circulation to the brain parenchyma. Genetically modified BECs enable protein secretion to the brain and provide a novel strategy to obtain brain uptake of otherwise BBB-impermeable proteins. However, secretion from BECs may be bidirectional, thereby leading to off-target secretion into the blood. This study investigates a vector strategy using the brain-specific AAV-BR1 viral vector to achieve specific transduction of BECs and to subsequently direct the recombinant proteins towards an abluminal secretion pathway, thereby increasing the release of recombinant proteins into the brain. METHODS: Different AAV-BR1 constructs, under the control of an endothelial-specific promoter (C-Ocln), were designed to include a fragment of the platelet-derived growth factor subunit B protein (PDGF-B) fused to the N-terminus of mCherry to promote abluminal secretion. Similar constructs using the more ubiquitous expressed CAG promoter were used for comparisons. The correct processing of the vector constructs was confirmed in vitro, including post-translational removal of the PDGF-B fragment by furin cleavage. AAV-BR1 vector constructs containing the C-Ocln promoter and the PDGF-B fragment were systemically administered in C57Bl/6J mice to evaluate abluminal secretion of mCherry to the brain parenchyma specifically by BECs. An in vitro BBB co-culture model based on primary mouse BECs and mixed glial cells was likewise used to evaluate if the PDGF-B fragment enhanced abluminal secretion of mCherry. RESULTS: The C-Ocln promoter exhibited low specificity to BECs, off-target transduction of neurons, and low transduction efficiency, which complicated the in vivo quantification of mCherry secretion into the brain parenchyma. Modelling the BBB in vitro showed higher mCherry secretion across the abluminal membrane when the PDGF-B fragment was included in the vector construct, especially in combination with the CAG promoter. CONCLUSION: Investigating abluminal secretion of mCherry was complicated in vivo by high off-target transduction of neurons, despite using the endothelial-specific promoter (C-Ocln). In vitro results, however, suggest that incorporating a PDGF-B fragment into the vector design targets the recombinant protein for abluminal secretion by BECs and facilitates increased protein delivery to the brain. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-025-00738-6.