RNA delivery to the corneal endothelium using charge-altering releasable transporters

RNA therapies hold tremendous promise for treating genetic eye diseases. However, their development is limited by the lack of non-viral delivery platforms that can target specific ocular cell types. Here, we describe a charge-altering releasable transporter (CART) that delivers RNA selectively to the corneal endothelium, a non-regenerative cell layer whose dysfunction underlies several blinding conditions. We characterize the safety of CART-RNA nanoparticles in mice and show that they facilitate delivery of diverse RNA cargoes to the corneal endothelium, including circular RNA and CRISPR/Cas9. We verify the feasibility of redosing these nanoparticles and apply them to achieve corneal gene editing. We further demonstrate CART transfection of corneal endothelial cells from a human donor in vitro and in a non-human primate in vivo, supporting translatability to patients. Our findings establish CARTs as a platform for non-viral gene delivery to the eye, with the potential to treat corneal dystrophies and other vision disorders. Overall design: RNA-seq of sorted cells from Ai9 mouse anterior segments without treatment or 1 week after intracameral injection of Cre mRNA with CART 8. RNA-seq of C57BL/6J mouse anterior segments 48 hours after intracameral injection of EGFP mRNA with or without CART 8.