Targeting genomic rearrangements in tumor cells through cas9-mediated insertion of a suicide gene

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

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BEING VIEWED BY OTHERS HIGH-EFFICIENCY TRANSGENE INTEGRATION BY HOMOLOGY-DIRECTED REPAIR IN HUMAN PRIMARY CELLS USING DNA-PKCS INHIBITION Article 03 August 2023 IN VIVO CRISPR/CAS9 TARGETING

OF FUSION ONCOGENES FOR SELECTIVE ELIMINATION OF CANCER CELLS Article Open access 08 October 2020 EFFICIENT VIRAL DELIVERY OF CAS9 INTO HUMAN SAFE HARBOR Article Open access 08 December

2020 REFERENCES * Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. _Cell_ 144, 646–674 (2011). Article  CAS  Google Scholar  * Yu, Y.P. et al. Novel fusion

transcripts associate with progressive prostate cancer. _Am. J. Pathol._ 184, 2840–2849 (2014). Article  CAS  Google Scholar  * 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  * Jinek, M. et

al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. _Science_ 337, 816–821 (2012). Article  CAS  Google Scholar  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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 

* 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). * Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. _Cell_ 100, 57–70 (2000). Article  CAS  Google Scholar  * 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  * Hsu, P.D. et al. DNA targeting specificity of

RNA-guided Cas9 nucleases. _Nat. Biotechnol._ 31, 827–832 (2013). Article  CAS  Google Scholar  * Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. _Science_ 339,

819–823 (2013). Article  CAS  Google Scholar  * Kozarsky, K.F. & Wilson, J.M. Gene therapy: adenovirus vectors. _Curr. Opin. Genet. Dev._ 3, 499–503 (1993). Article  CAS  Google Scholar

  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * Yu, G. et al. CSR1 suppresses tumor growth and metastasis of prostate cancer. _Am. J. Pathol._ 168, 597–607 (2006). Article  CAS 

Google Scholar  * 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  * 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  * Ren, B. et al. MCM7 amplification and overexpression are associated with prostate cancer progression. _Oncogene_ 25, 1090–1098 (2006). Article  CAS 

Google Scholar  * 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  * 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  * 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  * 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 * 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 * Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh,

Pennsylvania, USA Joel B Nelson * Department of Biostatistics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Silvia Liu & George Tseng Authors * Zhang-Hui

Chen View author publications You can also search for this author inPubMed Google Scholar * Yan P Yu View author publications You can also search for this author inPubMed Google Scholar *

Ze-Hua Zuo View author publications You can also search for this author inPubMed Google Scholar * Joel B Nelson View author publications You can also search for this author inPubMed Google

Scholar * George K Michalopoulos View author publications You can also search for this author inPubMed Google Scholar * Satdatshan Monga View author publications You can also search for this

author inPubMed Google Scholar * Silvia Liu View author publications You can also search for this author inPubMed Google Scholar * George Tseng View author publications You can also search

for this author inPubMed Google Scholar * Jian-Hua Luo View author publications You can also search for this author inPubMed 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 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: 30 June 2016 * Accepted: 08 March

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