Macrophage-specific in vivo RNA editing promotes phagocytosis and antitumor immunity in mice

Macrophages play a central role in antitumor immunity, making them an attractive target for gene therapy strategies. However, macrophages are difficult to transfect because of nucleic acid sensors that can trigger the degradation of foreign plasmid DNA. Here, we developed a macrophage-specific editing (MAGE) system by which compact plasmid DNA encoding a CasRx editor can be delivered to macrophages by a poly(β-amino ester) (PBAE) carrier to bypass the DNA sensor and enable RNA editing in vitro and in vivo. We identified a four-arm branched PBAE with 1-(2-aminoethyl)-4-methylpiperazine end-capping (PBAE29) that enables highly efficient macrophage transfection. PBAE29-mediated transfection of cultured macrophages stimulated less inflammatory cytokine production and inflammasome activation compared with traditional lipofectamine or electroporation-mediated plasmid delivery. Transfection efficiency was further improved by delivering CasRx by minicircle plasmid. The MAGE system incorporated a layer of carboxylated-mannan coating to target macrophage mannose receptors and a macrophage-specific promoter for enhanced selectivity. The delivery of CasRx with guide RNA targeting the transcripts for sialic acid-binding immunoglobulin similar to lectin 10 and signal regulatory protein alpha expression resulted in effective protein knockdown, improving macrophage phagocytosis. The MAGE system also showed efficacy in targeting macrophages in vivo, stimulating antitumor immune responses and reducing tumor volume in murine tumor models, including patient-derived pancreatic adenocarcinoma xenografts in humanized mice. In sum, the MAGE system presents a promising platform for in vivo macrophage-specific delivery of RNA editing tools that can be applied as a cancer therapy.