Targeted attenuation of liver fibrosis using microbubble-enhanced protease-activated receptor 1 chimeric antigen receptor T cells

BACKGROUND: Liver diseases account for a significant global mortality rate, with hepatic fibrosis representing a critical precursor to cirrhosis and liver cancer. Current therapeutic options remain limited, as no antifibrotic drugs have received FDA approval. In this study, we propose a novel CAR-T cell strategy targeting protease-activated receptor 1 (PAR1) on activated hepatic stellate cells (aHSCs) to combat liver fibrosis. METHODS: By engineering CAR-T cells specifically against PAR1, we aimed to selectively eliminate PAR1-expressing HSCs and thereby inhibit PAR1-dependent fibrogenesis. To enhance CAR-T cell delivery into the fibrotic liver microenvironment, we employed ultrasound-mediated delivery with microbubbles (USMB) in a Carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. FINDINGS: Our results demonstrated that PAR1CAR-T cells effectively eliminated PAR1-expressing HSCs in vitro. In animals, USMB significantly enhanced CAR-T cell penetration, migration, and intrahepatic retention within the fibrotic liver microenvironment. Blockade of PAR1 signaling suppressed HSC activation, attenuated fibrogenesis, and reduced fibrosis progression. Mechanistically, activation of the TGF-β/p-SMAD2/3 axis was accompanied by upregulation of PAR1 on activated HSCs, and this fibrogenic axis was attenuated by the targeted elimination of PAR1-expressing HSCs. INTERPRETATION: Collectively, USMB-mediated PAR1CAR-T cell therapy demonstrated potent antifibrotic efficacy by enhancing CAR-T cell delivery and intrahepatic retention in the liver, offering a promising antifibrotic immunotherapy approach for patients with liver fibrosis.