CAR Macrophages Engineered In Vivo for Attenuating Myocardial Ischemia-Reperfusion Injury.

Abstract

BACKGROUND Myocardial ischemia-reperfusion (I/R) injury induces myocardial fibrosis that compromises cardiac function and electrical conduction, yet current clinical options remain inadequate. To address this unmet need, we explored macrophage-targeted lipid nanoparticles (LNPs) encapsulating FAP CAR (FAP [fibroblast activation protein]-targeted chimeric antigen receptor) mRNA for in vivo generation of FAP CAR macrophages and evaluated their therapeutic potential in reducing myocardial fibrosis and improving cardiac function after myocardial I/R injury. METHODS We formulated 1,2-dioleoyl-sn-glycero-3-phospho-l-serine-doping ALC-0315 (an ionizable lipid) LNP to deliver FAP CAR mRNA to generate FAP CAR macrophages. The platform was first validated in vitro by assessing phagocytosis of FAP-overexpressing fibroblasts by these macrophages. For in vivo evaluation, C57BL/6J mice subjected to I/R injury received intravenous administration of PBS, control LNPs, or LNP-FAP CAR (LNPs encapsulating mRNA encoding a FAP-targeting CAR). Comprehensive analyses included tracking the biodistribution of the resultant FAP CAR macrophages, quantitative measurement of fibrosis reduction, assessment of cardiac function by echocardiography, and safety evaluations. RESULTS LNP-FAP CAR successfully generated functional FAP CAR macrophages that demonstrated phagocytosis ability toward FAP-positive fibroblasts in vitro. In vivo studies revealed that intravenous delivery of LNP-FAP CAR generated functional FAP CAR macrophages that selectively engaged and phagocytosed activated cardiac fibroblasts in I/R mouse hearts. This targeted cell clearance translated to a significant reduction in the number of activated cardiac fibroblasts and the extent of myocardial fibrosis, as well as marked improvement in cardiac function without detectable toxicities. Notably, these effects were achievable even when intervention was delayed for up to 2 weeks post-I/R. CONCLUSIONS Our study demonstrates that FAP CAR macrophages generated in vivo by LNP-FAP CAR treatment effectively mitigate cardiac fibrosis and improve heart function after I/R injury, with lasting benefits and no observed toxicity. This safe and adaptable platform offers a promising treatment strategy for myocardial I/R injury and holds potential for treating other fibrotic heart diseases.