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. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵$ Co-Corresponding Authors: Nicolas Marzolini: Nicolas.Marzolini{at}pennmedicine.upenn.edu, Taylor V. Brysgel: Taylor.Brysgel{at}pennmedicine.upenn.edu, Jacob S. Brenner: Jacob.Brenner{at}pennmedicine.upenn.edu, Sahily Reyes-Esteves: Sahily.Reyes-Esteves{at}pennmedicine.upenn.edu, Vladimir R. Muzykantov: muzykant{at}pennmedicine.upenn.edu Table of Contents: Attaching antibodies against endothelial cell surface proteins redirects the delivery and expression of DNA-lipid nanoparticles to organs of interest. Our targeted nanoparticles enable organ-selective DNA expression in the endothelium of the lungs, brain, or spleen, providing a therapeutic platform for dozens of endothelial-centric diseases. Figures 2 and 3 have been significantly revised; author affiliations updated; supplemental files updated; introduction and discussion sections updated