Modulation of miR‐23b Wnt/β‐catenin Axis Strengthens Endothelial Barrier Properties

ABSTRACT Blood‐brain barrier (BBB) disruption drives stroke and other CNS disorders pathology, yet restoring its integrity remains challenging. Using an unbiased anti‐miR lentiviral screen, miR‐23b was identified as a critical negative regulator of BBB integrity in brain endothelial cells (BECs). Targeted inhibition of miR‐23b using anti‐miR‐23b, validated by Wnt‐reporter, Wnt‐rescue, and eCLIP analysis, demonstrates that BEC miR‐23b silencing, enhances junctional protein expression and strengthens barrier function while suppressing transcellular transport. Global multi‐omics analysis reveals that miR‐23b acts as a robust driver of angiogenesis‐specific genes and proteins. Conversely, anti‐miR‐23b induces the expression of factors essential for BBB stabilization and repair. Notably, anti‐miR‐23b enhances these protective barrier properties through a multi‐faceted regulation of Wnt/β‐catenin, TGF‐β, Notch, and VEGF signaling mechanisms. Using a 3D microfluidic platform, anti‐miR‐23b was shown to foster BBB repair by accelerating vessel maturation and enhancing resilience against ischemic injury. Proof‐of‐concept studies show that BEC‐specific AAVBR1‐anti‐miR‐23b gene therapy reinforces BBB tight junctions, reduces BBB leakage, and improves outcome measures in a transient middle cerebral artery occlusion (t‐MCAO) stroke model. Thus, miR‐23b is a critical regulator of cerebrovascular integrity, positioning anti‐miR‐23b as a promising RNA‐based therapy to enhance BBB stability and repair in stroke and other CNS disorders.