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Advanced Science

Advanced Science

IF: 14.3
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Modular Calcium-Responsive and CD9-Targeted Phospholipase System Enhancing Endosomal Escape for DNA Delivery

Published:25 February 2025 DOI: 10.1002/advs.202410815 PMID: 39998318
Alexander Klipp, Christina Greitens, David Scherer, Alexander Elsener, Jean-Christophe Leroux, Michael Burger

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.

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