Abstract
BackgroundThe pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development.MethodsWe applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis.ResultsBBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis.ConclusionsHuman BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.
Highlights
The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized
Our results demonstrate that BBB organoid arrays can be combined with Clustered regularly interspaced short palindromic repeats (CRISPR) gene editing to interrogate the molecular mechanisms of transcytosis across the human BBB
0 USnctrraematbelded cTofnrteroclepCtolar tKhOrin HCCKavOeolin-1 KO Fig. 5 CRISPR/Cas9 gene editing in blood–brain barrier organoids does not disrupt barrier function. a Representative confocal images of blood– brain barrier organoids assembled with hCMEC/D3 Cas9/Scrambled gRNA control or knockout cells
Summary
The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. The diversity of shuttle formats (e.g. bispecific antibodies [1], single domain antibodies [6,7,8,9], Fc-engineered binders [3]) together with different receptor targets in brain endothelial cells (e.g. transferrin receptor [2, 10,11,12], insulin receptor [13], CD98 [14], TMEM30 [15]) expand the design space for successful brain delivery in a combinatorial manner Despite these rapid advances in protein delivery technologies to the central nervous system (CNS), the understanding of the precise molecular mechanisms of receptor-mediated antibody transport across the BBB is lagging. While these landmark efforts identified novel regulators for specific pathways, such as Mfsd2a [20] and ALPL [23], in vivo studies do not enable sufficiently high throughput and are not optimal to perform unbiased system-level analysis of transport mechanisms
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