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Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy

Nature Nanotechnology volume 17, pages 777–787 (2022)Cite this article

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Abstract

Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumours for efficient cancer therapy remains challenging. Here we targeted tumour tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA (siFAK + CRISPR-LNPs) to enable tumour delivery and enhance gene-editing efficacy. We show that gene editing was enhanced >10-fold in tumour spheroids due to increased cellular uptake and tumour penetration of nanoparticles mediated by FAK-knockdown. siFAK + CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumour growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumour tissue can enhance gene editing in tumours, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing.

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Scheme 1
Fig. 1: FAK knockdown enhances LNP-mediated mRNA delivery and CRISPR gene editing.
Fig. 2: FAK-knockdown enhances the endocytosis of siFAK + CRISPR-LNPs through dynamic alteration of the contraction force and cell membrane tension.
Fig. 3: siFAK + CRISPR-PD-L1-LNPs targeted tumour stiffness and PD-L1 to inhibit xenograft tumour growth.
Fig. 4: siFAK + CRISPR-LNPs enabled enhancement of gene editing though decreasing tumour stiffness in an aggressive, genetically engineered liver cancer model.
Fig. 5: Systemic administration of siFAK + CRISPR-PD-L1-LNPs significantly extended survival of mice bearing aggressive, MYC-driven cancer.

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All data that support the plots within this paper and other findings of this study are shown in the figures and are available from the corresponding author upon reasonable request.

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Acknowledgements

D.J.S. acknowledges support from the Cancer Prevention and Research Institute of Texas (CPRIT) (RP190251), the National Institutes of Health (NIH) (R01 EB025192-01A1, R01 CA269787-01), the American Cancer Society (ACS) (RSG-17-012-01) and the Cystic Fibrosis Foundation (CFF) (SIEGWA18XX0). H.Z. acknowledges support from the NIH (R01 DK111588, R01 DK125396, R01 CA251928), the Moody Medical Research Institute and an Emerging Leader Award from the Mark Foundation for Cancer Research (#21-003-ELA). We also acknowledge the UTSW Tissue Resource (National Cancer Institute (5P30CA142543)) and the Moody Foundation Flow Cytometry Facility. T.W. acknowledges a CPRIT Training Grant (RP160157). L.T.J. acknowledges the Pharma Foundation. We are also very grateful to Z. S. Guo (University of Pittsburgh) for sharing ID8-Luc cells with us.

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Authors and Affiliations

  1. Department of Biochemistry, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Di Zhang, Xueliang Yu, Tuo Wei, Lukas Farbiak, Lindsay T. Johnson & Daniel J. Siegwart

  2. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Guoxun Wang

  3. Department of Bioengineering, University of Texas at Arlington, Arlington, TX, USA

    Alan Mark Taylor, Jiazhu Xu & Yi Hong

  4. Children’s Research Institute, Departments of Pediatrics and Internal Medicine, Center for Regenerative Science and Medicine, Children’s Research Institute Mouse Genome Engineering Core, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Hao Zhu

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Contributions

D.Z. and D.J.S. designed the research. D.Z. designed and performed the experiments. G.W., X.Y., T.W., L.F. and L.T.J., performed the experiments. D.Z., A.M.T., J.X. and Y.H. performed the experiments and data analyses related to mechanical testing. All the authors were involved in the data analyses. D.Z. and D.J.S. wrote the manuscript. H.Z. and all authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Daniel J. Siegwart.

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Competing interests

D.J.S. is a co-founder and consultant of ReCode Therapeutics, which has licensed intellectual property from UT Southwestern. H.Z. has a sponsored research agreement with Alnylam Pharmaceuticals, consults for Flagship Pioneering and serves on the Scientific Advisory Board of Ubiquitix. H.Z.’s interests are not directly related to the contents of this paper. The other authors declare no competing interests.

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Zhang, D., Wang, G., Yu, X. et al. Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy. Nat. Nanotechnol. 17, 777–787 (2022). https://doi.org/10.1038/s41565-022-01122-3

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