mRNA-Laden Lipid-Nanoparticle-Enabled <i>in Situ</i> CAR-Macrophage Engineering for the Eradication of Multidrug-Resistant Bacteria in a Sepsis Mouse Model

Abstract

Sepsis, which is the most severe clinical manifestation of acute infection and has a mortality rate higher than that of cancer, represents a significant global public health burden. Persistent methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infection and further host immune paralysis are the leading causes of sepsis-associated death, but limited clinical interventions that target sepsis have failed to effectively restore immune homeostasis to enable complete eradication of MRSA. To restimulate anti-MRSA innate immunity, we developed CRV peptide-modified lipid nanoparticles (CRV/LNP-RNAs) for transient <i>in situ</i> programming of macrophages (MΦs). The CRV/LNP-RNAs enabled the delivery of MRSA-targeted chimeric antigen receptor (CAR) mRNA (SasA-CAR mRNA) and CASP11 (a key MRSA intracellular evasion target) siRNA to MΦs <i>in situ</i>, yielding CAR-MΦs with boosted bactericidal potency. Specifically, our results demonstrated that the engineered MΦs could efficiently phagocytose and digest MRSA intracellularly, preventing immune evasion by the "superbug" MRSA. Our findings highlight the potential of nanoparticle-enabled <i>in vivo</i> generation of CAR-MΦs as a therapeutic platform for multidrug-resistant (MDR) bacterial infections and should be confirmed in clinical trials.