Impact of ASO conjugation and receptor binding affinity on intracellular transport of mono- and bispecific TfR- and CD98-BrainshuttleTM variants.

Abstract

Antisense oligonucleotides (ASOs) represent a promising therapeutic modality for central nervous system (CNS) disorders, offering highly specific modulation of gene expression. However, their clinical utility is severely limited by their inability to cross the blood-brain barrier (BBB), necessitating effective shuttling strategies. While transferrin receptor (TfR1)-mediated shuttling has shown therapeutic promise, the fundamental mechanisms governing the delivery of antibody-ASO conjugates across the BBB remain poorly understood. This study directly addresses this critical knowledge gap by establishing a mechanistic understanding of how the ASO cargo impacts major cellular interactions during the Brainshuttle^TM-mediated transport across the BBB. Using a panel of advanced in vitro assays developed specifically for this purpose, including quantitative transcytosis, detailed imaging-based intracellular trafficking, and binding assays with brain endothelial cells (BECs), the shuttling process was systematically investigated. We demonstrate that ASO conjugation profoundly alters the cellular fate of the Brainshuttle^TM. Specifically, conjugation increased the binding to BECs of low-affinity TfR1 shuttles via avidity effects while paradoxically reducing the binding strength of high-affinity shuttles. Functional assays confirmed the biological activity of the delivered ASOs; however, transcytosis of high-to-moderate affinity binders across the BBB model was significantly delayed upon ASO conjugation. Building on these mechanistic insights, we engineered TfR1 Brainshuttles^TM with optimized affinity and explored the shuttling potential of an alternative BBB receptor, CD98hc. These efforts culminated in the development of a novel bispecific Brainshuttle^TM targeting both CD98hc and TfR1. This dual-targeting strategy exploits distinct and potentially non-competing trafficking pathways to overcome ASO-induced delays and significantly enhance in vitro transcytosis efficiency. The in vitro findings in this study underscore the necessity of mechanism-driven design to overcome ASO-induced limitations in delivery across the BBB. The bispecific CD98/TfR1 approach presented here provides a promising new strategy for maximizing delivery efficiency and enabling more effective therapeutic outcomes for CNS diseases.