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.

  • Article
  • Published:

Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene

Nature Biotechnology volume 35, pages 543–550 (2017)Cite this article

Subjects

Abstract

Specifically targeting genomic rearrangements and mutations in tumor cells remains an elusive goal in cancer therapy. Here, we used Cas9-based genome editing to introduce the gene encoding the prodrug-converting enzyme herpes simplex virus type 1 thymidine kinase (HSV1-tk) into the genomes of cancer cells carrying unique sequences resulting from genome rearrangements. Specifically, we targeted the breakpoints of TMEM135–CCDC67 and MAN2A1–FER fusions in human prostate cancer or hepatocellular carcinoma cells in vitro and in mouse xenografts. We designed one adenovirus to deliver the nickase Cas9D10A and guide RNAs targeting the breakpoint sequences, and another to deliver an EGFP-HSV1-tk construct flanked by sequences homologous to those surrounding the breakpoint. Infection with both viruses resulted in breakpoint-dependent expression of EGFP-tk and ganciclovir-mediated apoptosis. When mouse xenografts were treated with adenoviruses and ganciclovir, all animals showed decreased tumor burden and no mortality during the study. Thus, Cas9-mediated suicide-gene insertion may be a viable genotype-specific cancer therapy.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic of strategy to introduce EGFP-tk into the breakpoint of the TMEM135–CCDC67 fusion gene.
Figure 2: EGFP-tk integration and expression in cells expressing the TMEM135–CCDC67-fusion breakpoint transcript.
Figure 3: Treatment with the nucleotide analog ganciclovir kills cancer cells expressing EGFP-tk.
Figure 4: Treatment with ganciclovir induces partial remission of xenografted prostate cancers in SCID mice.
Figure 5: Genome therapy targeting at the MAN2A1–FER breakpoint.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Yu, Y.P. et al. Novel fusion transcripts associate with progressive prostate cancer. Am. J. Pathol. 184, 2840–2849 (2014).

    Article  CAS  Google Scholar 

  3. Mojica, F.J., Díez-Villaseñor, C., García-Martínez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Esvelt, K.M., Smidler, A.L., Catteruccia, F. & Church, G.M. Concerning RNA-guided gene drives for the alteration of wild populations. eLife 3, 03401 (2014).

    Article  Google Scholar 

  6. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  7. Smith, K.O., Galloway, K.S., Kennell, W.L., Ogilvie, K.K. & Radatus, B.K. A new nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxyl]methyl]guanine, highly active in vitro against herpes simplex virus types 1 and 2. Antimicrob. Agents Chemother. 22, 55–61 (1982).

    Article  CAS  Google Scholar 

  8. Van Rompay, A.R., Johansson, M. & Karlsson, A. Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases. Pharmacol. Ther. 87, 189–198 (2000).

    Article  CAS  Google Scholar 

  9. Yu, Y.P. et al. Genomic copy number variations in the genomes of leukocytes predict prostate cancer clinical outcomes. PLoS One 10, e0135982 (2015).

    Article  Google Scholar 

  10. Luo, J.H. et al. Discovery and classification of fusion transcripts in prostate cancer and normal prostate tissue. Am. J. Pathol. 185, 1834–1845 (2015).

    Article  CAS  Google Scholar 

  11. Ohnuki, Y., Marnell, M.M., Babcock, M.S., Lechner, J.F. & Kaighn, M.E. Chromosomal analysis of human prostatic adenocarcinoma cell lines. Cancer Res. 40, 524–534 (1980).

    CAS  PubMed  Google Scholar 

  12. Bernardino, J. et al. Characterization of chromosome changes in two human prostatic carcinoma cell lines (PC-3 and DU145) using chromosome painting and comparative genomic hybridization. Cancer Genet. Cytogenet. 96, 123–128 (1997).

    Article  CAS  Google Scholar 

  13. Chen, Z.H. et al. MAN2A1-FER fusion gene is expressed by human liver and other tumor types and has oncogenic activity in mice. Gastroenterology http://dx.doi.org/10.1053/j.gastro.2016.12.036 (2017).

  14. Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57–70 (2000).

    Article  CAS  Google Scholar 

  15. Yu, C. et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16, 142–147 (2015).

    Article  CAS  Google Scholar 

  16. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Kozarsky, K.F. & Wilson, J.M. Gene therapy: adenovirus vectors. Curr. Opin. Genet. Dev. 3, 499–503 (1993).

    Article  CAS  Google Scholar 

  19. Anderson, R.D., Haskell, R.E., Xia, H., Roessler, B.J. & Davidson, B.L. A simple method for the rapid generation of recombinant adenovirus vectors. Gene Ther. 7, 1034–1038 (2000).

    Article  CAS  Google Scholar 

  20. Wang, H. et al. p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3. J. Biol. Chem. 287, 16890–16902 (2012).

    Article  CAS  Google Scholar 

  21. Zhu, Z.H. et al. Integrin alpha 7 interacts with high temperature requirement A2 (HtrA2) to induce prostate cancer cell death. Am. J. Pathol. 177, 1176–1186 (2010).

    Article  CAS  Google Scholar 

  22. Luo, K.L., Luo, J.H. & Yu, Y.P. (−)-Epigallocatechin-3-gallate induces Du145 prostate cancer cell death via downregulation of inhibitor of DNA binding 2, a dominant negative helix-loop-helix protein. Cancer Sci. 101, 707–712 (2010).

    Article  CAS  Google Scholar 

  23. Han, Y.C. et al. Interaction of integrin-linked kinase and miniature chromosome maintenance 7-mediating integrin alpha7 induced cell growth suppression. Cancer Res. 70, 4375–4384 (2010).

    Article  CAS  Google Scholar 

  24. Zhu, Z.H., Yu, Y.P., Shi, Y.K., Nelson, J.B. & Luo, J.H. CSR1 induces cell death through inactivation of CPSF3. Oncogene 28, 41–51 (2009).

    Article  CAS  Google Scholar 

  25. Shi, Y.K., Yu, Y.P., Tseng, G.C. & Luo, J.H. Inhibition of prostate cancer growth and metastasis using small interference RNA specific for minichromosome complex maintenance component 7. Cancer Gene Ther. 17, 694–699 (2010).

    Article  CAS  Google Scholar 

  26. Yu, Y.P. et al. Glutathione peroxidase 3, deleted or methylated in prostate cancer, suppresses prostate cancer growth and metastasis. Cancer Res. 67, 8043–8050 (2007).

    Article  CAS  Google Scholar 

  27. Ren, B. et al. Analysis of integrin alpha7 mutations in prostate cancer, liver cancer, glioblastoma multiforme, and leiomyosarcoma. J. Natl. Cancer Inst. 99, 868–880 (2007).

    Article  CAS  Google Scholar 

  28. Yu, G. et al. CSR1 suppresses tumor growth and metastasis of prostate cancer. Am. J. Pathol. 168, 597–607 (2006).

    Article  CAS  Google Scholar 

  29. Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).

    Article  CAS  Google Scholar 

  30. Han, Y.C. et al. Metallothionein 1 h tumour suppressor activity in prostate cancer is mediated by euchromatin methyltransferase 1. J. Pathol. 230, 184–193 (2013).

    Article  CAS  Google Scholar 

  31. Ren, B. et al. MCM7 amplification and overexpression are associated with prostate cancer progression. Oncogene 25, 1090–1098 (2006).

    Article  CAS  Google Scholar 

  32. Jing, L. et al. Expression of myopodin induces suppression of tumor growth and metastasis. Am. J. Pathol. 164, 1799–1806 (2004).

    Article  CAS  Google Scholar 

  33. Demetris, A.J., Seaberg, E.C., Wennerberg, A., Ionellie, J. & Michalopoulos, G. Ductular reaction after submassive necrosis in humans: special emphasis on analysis of ductular hepatocytes. Am. J. Pathol. 149, 439–448 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Yu, Y.P. et al. Whole-genome methylation sequencing reveals distinct impact of differential methylations on gene transcription in prostate cancer. Am. J. Pathol. 183, 1960–1970 (2013).

    Article  CAS  Google Scholar 

  35. Lin, F. et al. Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers. Am. J. Pathol. 159, 1603–1612 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Zheng for technical support. This work was supported by grants from the National Cancer Institute to JHL (RO1 CA098249 to J.-H.L.), the Department of Defense (W81XWH-16-1-0364) to J.-H.L. and the University of Pittsburgh Cancer Institute to J.-H.L., G.K.M. and J.B.N.

Author information

Authors and Affiliations

  1. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Zhang-Hui Chen, Yan P Yu, Ze-Hua Zuo, George K Michalopoulos, Satdatshan Monga & Jian-Hua Luo

  2. Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Joel B Nelson

  3. Department of Biostatistics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Silvia Liu & George Tseng

Authors
  1. Zhang-Hui Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yan P Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ze-Hua Zuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joel B Nelson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. George K Michalopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Satdatshan Monga
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Silvia Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. George Tseng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jian-Hua Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.-H.L. and Y.P.Y. conceived the concept of the project and devised the research strategy. Z.-H.C. and Z.-H.Z. performed most experiments, S.M. provided materials, G.K.M. and J.B.N. provided expertise and advice on the biology of and therapies for liver cancer and prostate cancer. S.L. and G.T. performed biostatistics and bioinformatics analyses.

Corresponding author

Correspondence to Jian-Hua Luo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Tables 1–7 (PDF 1593 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, ZH., Yu, Y., Zuo, ZH. et al. Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene. Nat Biotechnol 35, 543–550 (2017). https://doi.org/10.1038/nbt.3843

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3843

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research