Engineered nucleocytosolic vehicles for loading of programmable editors

  • Cell. 2025 May 15;188(10):2637-2655.e31. doi: 10.1016/j.cell.2025.03.015.
Julian Geilenkeuser  1 ,  Niklas Armbrust  1 ,  Emily Steinmaßl  1 ,  Samuel W Du  2 ,  Sebastian Schmidt  3 ,  Eva Maria Hildegard Binder  1 ,  Yuchun Li  1 ,  Niklas Wilhelm Warsing  1 ,  Stephanie Victoria Wendel  1 ,  Florian von der Linde  1 ,  Elisa Marie Schiele  1 ,  Xiya Niu  4 ,  Luisa Stroppel  1 ,  Oleksandr Berezin  1 ,  Tobias Heinrich Santl  1 ,  Tanja Orschmann  5 ,  Keith Nelson  1 ,  Christoph Gruber  6 ,  Grazyna Palczewska  7 ,  Carolline Rodrigues Menezes  2 ,  Eleonora Risaliti  2 ,  Zachary J Engfer  2 ,  Naile Koleci  8 ,  Andrea Schmidts  8 ,  Arie Geerlof  9 ,  Krzysztof Palczewski  10 ,  Gil Gregor Westmeyer  11 ,  Dong-Jiunn Jeffery Truong  12
Affiliations
  • 1. Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany; Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich, Munich, Germany.
  • 2. Gavin Herbert Eye Institute - Center for Translational Vision Research, Department of Ophthalmology, University of California, Irvine, Irvine, CA, USA; Department of Physiology & Biophysics, University of California, Irvine, Irvine, CA, USA.
  • 3. Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany; Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich, Munich, Germany; Institute of Developmental Genetics, Helmholtz Munich, Neuherberg, Germany.
  • 4. Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany.
  • 5. Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany; Institute of Developmental Genetics, Helmholtz Munich, Neuherberg, Germany.
  • 6. Institute of Developmental Genetics, Helmholtz Munich, Neuherberg, Germany.
  • 7. Gavin Herbert Eye Institute - Center for Translational Vision Research, Department of Ophthalmology, University of California, Irvine, Irvine, CA, USA.
  • 8. Department of Medicine III: Hematology/Oncology, Klinikum rechts der Isar of the Technical University of Munich, TUM School of Medicine and Health, Munich, Germany.
  • 9. Institute of Structural Biology, Helmholtz Munich, Neuherberg, Germany.
  • 10. Gavin Herbert Eye Institute - Center for Translational Vision Research, Department of Ophthalmology, University of California, Irvine, Irvine, CA, USA; Department of Physiology & Biophysics, University of California, Irvine, Irvine, CA, USA; Department of Chemistry, University of California, Irvine, Irvine, CA, USA; Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA.
  • 11. Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany; Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich, Munich, Germany. Electronic address: [email protected].
  • 12. Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany; Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich, Munich, Germany. Electronic address: [email protected].
PMID: 40209705 DOI: 10.1016/j.cell.2025.03.015
Abstract

Advanced Gene Editing methods have accelerated biomedical discovery and hold great therapeutic promise, but safe and efficient delivery of gene editors remains challenging. In this study, we present a virus-like particle (VLP) system featuring nucleocytosolic shuttling vehicles that retrieve pre-assembled Cas-effectors via aptamer-tagged guide RNAs. This approach ensures preferential loading of fully assembled editor ribonucleoproteins (RNPs) and enhances the efficacy of prime editing, base editing, trans-activators, and Nuclease activity coupled to homology-directed repair in multiple immortalized, primary, stem cell, and stem-cell-derived cell types. We also achieve additional protection of inherently unstable prime editing guide RNAs (pegRNAs) by shielding the 3'-exposed end with Csy4/Cas6f, further enhancing editing performance. Furthermore, we identify a minimal set of packaging and budding modules that can serve as a platform for bottom-up engineering of enveloped delivery vehicles. Notably, our system demonstrates superior per-VLP editing efficiency in primary T lymphocytes and two mouse models of inherited retinal disease, highlighting its therapeutic potential.

Keywords
VLPs; base editing; cell therapy; gene delivery; gene therapy; genome editing; in vivo delivery; prime editing; virus-like particles.
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