Modular Calcium‐Responsive and CD9‐Targeted Phospholipase System Enhancing Endosomal Escape for DNA Delivery

Abstract Gene delivery systems must overcome multiple barriers, with endosomal escape representing a prominent obstacle. We have previously shown that a bacterial phospholipase C (PLC) enabled endosomal escape of a non‐viral protein‐based DNA delivery system termed TFAMoplex. Building upon this, this work introduces a calcium‐responsive system designed to enhance endosomal escape through non‐covalent capturing of PLC to the TFAMoplex followed by its release within endosomes and nanobody‐mediated targeting to the endosomal membrane. This approach leads to improved TFAMoplexes enabling transfection of HeLa cells in full serum with a half maximal effective concentration (EC₅₀) of less than 200 ng DNA per mL serum, using only 5 nM PLC. Particularly, the modular capture, release and targeting system could potentially be adapted to other delivery agents previously constrained by poor endosomal escape. These findings present a promising strategy to achieve efficient endosomal escape, offering prospects for improved delivery of macromolecules, in particular nucleic acids.