mRNA/LNP-mediated expression of a ligand-independent Tie2 pathway activator, COMP-Tie2*, enables sustained endothelial stabilization

Abstract

Activation of the angiopoietin/Tie2 signaling axis is a validated strategy for endothelial stabilization. However, therapeutic Tie2 activation via angiopoietin-1 (Ang1) or Ang1-derived agonists is intrinsically limited due to several mechanism including ligand instability, competitive Ang2 inhibition, Tie2 ectodomain cleavage, VE-PTP-mediated Tie2 dephosphorylation, and ligand-induced receptor degradation. All these inhibitory mechanisms restrict both magnitude as well as duration of therapeutic Tie2 signaling, particularly under inflammatory conditions such as acute respiratory distress syndrome (ARDS) or sepsis. Here, we describe a novel ligand-independent strategy for sustained Tie2 activation based on engineered membrane-anchored Tie2 variants. By combining intracellular Tie2 kinase domains harboring naturally occurring autoactivating mutations with heterologous membrane-targeting domains, we generated truncated, constitutively active Tie2 variants that bypass extracellular ligand regulation entirely. mRNA-mediated expression of these constructs induced robust and persistent Tie2 autophosphorylation and downstream Akt-FOXO1 signaling in pulmonary endothelial cells, which exceeded COMP-Ang1-mediated activation in both strength and duration. Systemic administration of COMP-Tie2* mRNA using lung-targeted lipid nanoparticles resulted in selective pulmonary Tie2 receptor engagement in vivo. Collectively, these data establish LNP-mediated mRNA delivery of engineered recombinant receptors for intrinsic Tie2 activation as a promising novel approach for endothelial stabilization under inflammatory conditions.