LNP-mediated CRISPR gene editing of human vascular endothelial cells in 2D and 3D microfluidic cell culture systems
4 affiliations
- 1Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark
- 2Interdisciplinary Nanoscience Center (iNANO), Aarhus University, 8000 Aarhus, Denmark
- 3Department of Obstetrics and Gynecology, Aarhus University Hospital, 8200 Aarhus, Denmark
- 4Department for Biomedical Research, University of Bern, 3008 Bern, Switzerland
Abstract
Efficient and cell-type-specific delivery remains a major barrier to CRISPR-based genome editing in vascular endothelial cells. Here, we systematically evaluate lipid nanoparticle (LNP)-mediated mRNA and CRISPR delivery in human umbilical vein endothelial cells (HUVECs). While a conventional SM-102-based LNP formulation enables highly efficient mRNA delivery to both HEK293T cells and HUVECs, CRISPR-SpCas9 gene editing is observed exclusively in HEK293T cells, revealing strong cell-dependent limitations. We identify reduced SpCas9 mRNA translation as a key bottleneck in endothelial cells and demonstrate that rational modification of LNP lipid composition dramatically enhances SpCas9 protein expression and restores robust genome editing in HUVECs. Using the optimized LNP formulation, we achieve efficient editing of VEGFA and HIF1A in both 2D cultures and physiologically relevant 3D microfluidic endothelial systems mimicking flow conditions in vivo. Together, our results establish lipid composition as a critical determinant of endothelial CRISPR editing efficiency and provide a scalable, non-viral strategy for genome engineering in the vascular endothelium.
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BibTeX
@article{olsen2026lnpmediated,
title = {{LNP-mediated CRISPR gene editing of human vascular endothelial cells in 2D and 3D microfluidic cell culture systems}},
author = {Amalie Lykke Olsen and Camilla Blunk Vorsø Brandt and Rasmus Karred Larsen and Sofie Vestergaard Fonager and Judith Zubia-Aranburu and János Haskó and Rikke Bek Helmig and Søren Egedal Degn and Mingdong Dong and Niels Jessen and Anders Etzerodt and Robert Rieben and Lin Lin and Yonglun Luo},
journal = {Molecular therapy. Nucleic acids},
year = {2026},
month = oct,
doi = {10.1016/j.omtn.2026.103126},
url = {https://europepmc.org/article/PMC/PMC13717028},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - LNP-mediated CRISPR gene editing of human vascular endothelial cells in 2D and 3D microfluidic cell culture systems
AU - Amalie Lykke Olsen
AU - Camilla Blunk Vorsø Brandt
AU - Rasmus Karred Larsen
AU - Sofie Vestergaard Fonager
AU - Judith Zubia-Aranburu
AU - János Haskó
AU - Rikke Bek Helmig
AU - Søren Egedal Degn
AU - Mingdong Dong
AU - Niels Jessen
AU - Anders Etzerodt
AU - Robert Rieben
AU - Lin Lin
AU - Yonglun Luo
PY - 2026
DA - 2026/10/01
JO - Molecular therapy. Nucleic acids
DO - 10.1016/j.omtn.2026.103126
UR - https://europepmc.org/article/PMC/PMC13717028
AB - Efficient and cell-type-specific delivery remains a major barrier to CRISPR-based genome editing in vascular endothelial cells. Here, we systematically evaluate lipid nanoparticle (LNP)-mediated mRNA and CRISPR delivery in human umbilical vein endothelial cells (HUVECs). While a conventional SM-102-based LNP formulation enables highly efficient mRNA delivery to both HEK293T cells and HUVECs, CRISPR-SpCas9 gene editing is observed exclusively in HEK293T cells, revealing strong cell-dependent limitations. We identify reduced SpCas9 mRNA translation as a key bottleneck in endothelial cells and demonstrate that rational modification of LNP lipid composition dramatically enhances SpCas9 protein expression and restores robust genome editing in HUVECs. Using the optimized LNP formulation, we achieve efficient editing of VEGFA and HIF1A in both 2D cultures and physiologically relevant 3D microfluidic endothelial systems mimicking flow conditions in vivo. Together, our results establish lipid composition as a critical determinant of endothelial CRISPR editing efficiency and provide a scalable, non-viral strategy for genome engineering in the vascular endothelium.
SN - 2162-2531
ER -