Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

Data availability

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.

References

  1. Wang, H. X. et al. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chem. Rev. 117, 9874–9906 (2017).

    CAS  Article  Google Scholar 

  2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  3. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  Article  Google Scholar 

  4. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  5. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).

    CAS  Article  Google Scholar 

  6. Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

    CAS  Article  Google Scholar 

  7. Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.Nature 516, 423–427 (2014).

    CAS  Article  Google Scholar 

  8. Long, C. Z. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).

    CAS  Article  Google Scholar 

  9. Miller, J. B. et al. Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew. Chem. Int. Ed. 56, 1059–1063 (2017).

    CAS  Article  Google Scholar 

  10. Wei, T. et al. Delivery of tissue-targeted scalpels: opportunities and challenges for in vivo CRISPR/Cas-based genome editing. ACS Nano 14, 9243–9262 (2020).

    CAS  Article  Google Scholar 

  11. Huang, C. H., Lee, K. C. & Doudna, J. A. Applications of CRISPR-Cas enzymes in cancer therapeutics and detection. Trends Cancer 4, 499–512 (2018).

    CAS  Article  Google Scholar 

  12. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    CAS  Article  Google Scholar 

  13. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    CAS  Article  Google Scholar 

  14. Jiang, H. et al. Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nat. Med. 22, 851–860 (2016).

    CAS  Article  Google Scholar 

  15. Mohammadi, H. & Sahai, E. Mechanisms and impact of altered tumour mechanics. Nat. Cell Biol. 20, 766–774 (2018).

    CAS  Article  Google Scholar 

  16. Humphrey, J. D., Dufresne, E. R. & Schwartz, M. A. Mechanotransduction and extracellular matrix homeostasis. Nat. Rev. Mol. Cell Biol. 15, 802–812 (2014).

    CAS  Article  Google Scholar 

  17. Lampi, M. C. & Reinhart-King, C. A. Targeting extracellular matrix stiffness to attenuate disease: from molecular mechanisms to clinical trials. Sci. Trans. Med. 10, eaao0475 (2018).

    Article  CAS  Google Scholar 

  18. Seong, J., Wang, N. & Wang, Y. Mechanotransduction at focal adhesions: from physiology to cancer development. J. Cell. Mol. Med. 17, 597–604 (2013).

    CAS  Article  Google Scholar 

  19. Serrels, A. et al. Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity. Cell 163, 160–173 (2015).

    CAS  Article  Google Scholar 

  20. Casey, S. C. et al. MYC regulates the antitumor immune response through CD47 and PD-L1. Science 352, 227–231 (2016).

    CAS  Article  Google Scholar 

  21. Topalian, S. L., Drake, C. G. & Pardoll, D. M. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr. Opin. Immunol. 24, 207–212 (2012).

    CAS  Article  Google Scholar 

  22. Zhou, K. et al. Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl Acad. Sci. USA 113, 520–525 (2016).

    CAS  Article  Google Scholar 

  23. Cheng, Q. et al. Dendrimer-based lipid nanoparticles deliver therapeutic FAH mRNA to normalize liver function and extend survival in a mouse model of hepatorenal tyrosinemia type I. Adv. Mater. 30, e1805308 (2018).

    Article  CAS  Google Scholar 

  24. Cheng, Q. et al. Selective ORgan Targeting (SORT) nanoparticles for tissue specific mRNA delivery and CRISPR/Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020).

    CAS  Article  Google Scholar 

  25. Ball, R. L., Hajj, K. A., Vizelman, J., Bajaj, P. & Whitehead, K. A. Lipid nanoparticle formulations for enhanced co-delivery of siRNA and mRNA. Nano Lett. 18, 3814–3822 (2018).

    CAS  Article  Google Scholar 

  26. Patel, S. et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA. Nat. Commun. 11, 983 (2020).

    CAS  Article  Google Scholar 

  27. Abumanhal-Masarweh, H. et al. Tailoring the lipid composition of nanoparticles modulates their cellular uptake and affects the viability of triple negative breast cancer cells. J. Control. Release 307, 331–341 (2019).

    CAS  Article  Google Scholar 

  28. Wei, T., Cheng, Q., Min, Y.-L., Olson, E. N. & Siegwart, D. J. Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing. Nat. Commun. 11, 3232 (2020).

    CAS  Article  Google Scholar 

  29. Liu, S. et al. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing. Nat. Mater. 20, 701–710 (2021).

    CAS  Article  Google Scholar 

  30. Lee, S. M. et al. A systematic study of unsaturation in lipid nanoparticles leads to improved mRNA transfection in vivo. Angew. Chem. Int. Ed. 60, 5848–5853 (2021).

    CAS  Article  Google Scholar 

  31. Mehta, G., Hsiao, A. Y., Ingram, M., Luker, G. D. & Takayama, S. Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J. Control. Release 164, 192–204 (2012).

    CAS  Article  Google Scholar 

  32. Laklai, H. et al. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression. Nat. Med. 22, 497–505 (2016).

    CAS  Article  Google Scholar 

  33. Kaksonen, M. & Roux, A. Mechanisms of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 19, 313–326 (2018).

    CAS  Article  Google Scholar 

  34. Echarri, A. & Del Pozo, M. A. Caveolae–mechanosensitive membrane invaginations linked to actin filaments. J. Cell Sci. 128, 2747–2758 (2015).

    CAS  Google Scholar 

  35. Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).

    CAS  Article  Google Scholar 

  36. Kraning-Rush, C. M., Califano, J. P. & Reinhart-King, C. A. Cellular traction stresses increase with increasing metastatic potential. PLoS ONE 7, e32572 (2012).

    CAS  Article  Google Scholar 

  37. Chaudhuri, O. et al. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nat. Mater. 13, 970–978 (2014).

    CAS  Article  Google Scholar 

  38. Hoadley, K. A. et al. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin. Cell 158, 929–944 (2014).

    CAS  Article  Google Scholar 

  39. Stokes, J. B. et al. Inhibition of focal adhesion kinase by PF-562,271 inhibits the growth and metastasis of pancreatic cancer concomitant with altering the tumor microenvironment. Mol. Cancer Ther. 10, 2135–2145 (2011).

    CAS  Article  Google Scholar 

  40. Shachaf, C. M. et al. MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature 431, 1112–1117 (2004).

    CAS  Article  Google Scholar 

  41. Zheng, K., Cubero, F. J. & Nevzorova, Y. A. c-MYC making liver sick: role of c-MYC in hepatic cell function, homeostasis and disease. Genes 8, 123 (2017).

    Article  CAS  Google Scholar 

  42. Egeblad, M., Rasch, M. G. & Weaver, V. M. Dynamic interplay between the collagen scaffold and tumor evolution. Curr. Opin. Cell Biol. 22, 697–706 (2010).

    CAS  Article  Google Scholar 

  43. Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005).

    CAS  Article  Google Scholar 

  44. Fourcade, J. et al. CD8+ T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1. Cancer Res. 72, 887–896 (2012).

    CAS  Article  Google Scholar 

  45. Schreiber, R. D., Old, L. J. & Smyth, M. J. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 331, 1565–1570 (2011).

    CAS  Article  Google Scholar 

  46. Sica, A. & Mantovani, A. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 122, 787–795 (2012).

    CAS  Article  Google Scholar 

  47. Naito, Y. et al. CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer Res. 58, 3491–3494 (1998).

    CAS  Google Scholar 

  48. Zhang, S. et al. Knockdown of anillin actin binding protein blocks cytokinesis in hepatocytes and reduces liver tumor development in mice without affecting regeneration. Gastroenterology 154, 1421–1434 (2018).

    CAS  Article  Google Scholar 

  49. Miller, J. B. & Siegwart, D. J. Design of synthetic materials for intracellular delivery of RNAs: from siRNA-mediated gene silencing to CRISPR/Cas gene editing. Nano Res. 11, 5310–5337 (2018).

    CAS  Article  Google Scholar 

  50. Wu, S. Y., Lopez-Berestein, G., Calin, G. A. & Sood, A. K. RNAi therapies: drugging the undruggable. Sci. Trans. Med 6, 240–247 (2014).

    Google Scholar 

  51. Cox, A. D. & Der, C. J. Ras history: the saga continues. Small GTPases 1, 2–27 (2010).

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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.

Ethics declarations

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.

Peer review

Peer review information

Nature Nanotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplemental text, methods, Figs. 1–33, Tables 1 and 2, and source data.

Reporting Summary.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-022-01122-3

Search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research