A4 - AAV Vectors - Preclinical and Proof-of-Concept In-Vivo Studies (Excluding Non-Human Primates)
250: Addressing the Translational Potential of Neurotropic AAVs Using Functionally Mature Human Stem Cell-Derived Blood-Brain Barrier Organoids
Type: Oral Abstract Session
Presentation Details
Session Title: AAV Vectors - Preclinical and Proof-of-Concept: Technology Focus
Novel AAV9 engineered capsids have been selected for their superior ability to cross the blood-brain barrier (BBB) and diffuse into the brain parenchyma after intravascular administration. However, their behaviors have been assessed in mice up to non-human primates, but whether the same capacity remains unchanged in a human setting remains unknown. To address this, we established improved procedures to differentiate human pluripotent-stem cells (hPSCs) into endothelial, mural and astroglial cells by defining an original combination of small molecule cocktails with the expression of cell lineage-specific master regulators. To assemble the three cell types together we decided to generate organoids that recapitulate the spatial organization of the BBB on a transwell filter (fBBBOs). We demonstrated that fBBBOs display a tight and homogeneous endothelial cell layer with well-organized cell junctions clustering key endothelial cell junction components including Glut1 and Occludin. BBBOs developed high functional properties with high values of trans-epithelial electric resistance (TEER) and impermeability to high molecular mass dyes. Furthermore, we tested the fBBBO endothelial integrity by assessing any eventual crossing of AAV2 and lentiviruses (LVs) that notoriously are incapable to trespass this biological barrier in vivo. Indeed, the fBBBOs effectively blocked the diffusion of both LVs and AAV2. In contrast, the AAV-PHP.B was able to efficiently transverse the fBBBOs only when expressing its transcytosis receptor Ly6A. These results demonstrate that BBBOs have acquired a highly selective permeability. Altogether, the deployment of cell lineage master improves the consistency of cell differentiation and subsequent maturation state. Spatially-defined cultures of endothelial, mural and astroglial cells produced a robust in vitro neurovascular model with long-lasting stability and functions. Human fBBBOs offer a neat system for testing the translational potential of new brain shuttling agents, including the new AAV engineered variants exploited in animals or in vitro.
Plain Language Summary
We generated a new in vitro model of the blood-brain barrier (BBB) by differentiating human stem cells into endothelial, mural and glial cells and combining them into organoids. BBB organoids develop the main functional properties of the native human structure with a tight endothelial layer impermeable to the crossing of molecules. BBB organoids are precious models to validate the translational potential of viral and non-viral brain shuttles to effectively cross the human BBB after intravascular delivery.
Angelo Iannielli1, Martina Greco1, Serena G. Giannelli1, Mirko Luoni1,2, Benjamin E. Deverman3, Vania Broccoli1,2
1Ospedale San Raffaele, Milan, Italy,2Institute of Neuroscience, CNR, Milan, Italy,3Broad Institute, Cambridge, MA"