Targeting DNA‐LNPs to Endothelial Cells Improves Expression Magnitude, Duration, and Specificity

ABSTRACT DNA‐lipid nanoparticles (DNA‐LNPs) loaded with inhibitors of the cGAS‐STING pathway enable safe and effective delivery of DNA in vivo. Herein, we report the first instances of extrahepatic DNA‐LNP targeting. DNA‐LNPs conjugated to antibodies against PECAM‐1 or VCAM‐1 target the endothelium of the lungs and brain/spleen, respectively. These LNPs drive robust transgene expression in their target organs, with greater magnitude and duration than untargeted LNPs. Lung specificity of PECAM‐targeted transgene expression increases over two weeks, resulting in markedly higher lung‐to‐liver expression ratios than our previous PECAM‐targeted mRNA‐LNPs. Off‐target liver DNA expression declines to undetectable levels but persists in the lungs, while mRNA expression uniformly decreases due to its short half‐life. We further improve this expression specificity by replacing full‐length antibodies with Fab fragments. Single‐cell analysis reveals a key mechanism underlying the improvements in organ‐specificity: target organ expression is dominated by long‐lived endothelial cells, while off‐target liver delivery and expression are in non‐endothelial cells with shorter half‐lives. Collectively, these studies demonstrate that targeted DNA‐LNPs achieve high levels of organ‐ and cell‐type‐specific transgene expression and thus provide a therapeutic platform for dozens of endothelial‐centric diseases.