Cancer is caused by a series of alterations in genome and epigenome and exists in multiple complex forms, making it difficult to be prevented and/or treated. Telomerase, an enzyme responsible for the maintenance of telomere, is silent in most normal somatic cells but activated in 90% of cancer cells, making it an excellent target for cancer therapy. Therefore, various telomerase activity inhibitors have been developed to treat cancer but all failed due to side effects. Here we acted oppositely to develop a cancer gene therapy named telomerase-activating gene expression (Tage) system by utilizing the telomerase activity in cancer cells. The Tage system consisted of an effector gene expression vector that carried a 3ʹ telomerase-recognizable stick end and an artificial transcription factor expression vector that could express dCas9-VP64 and an sgRNA targeting telomere repeat sequences. By using Cas9 as an effector gene, the Tage system effectively killed various cancer cells, including HepG2, HeLa, PANC-1, MDA-MB-453, A549, HT-29, SKOV-3, Hepa1-6, and RAW264.7, without affecting normal cells MRC-5, HL7702, and bone marrow mesenchymal stem cell (BMSC). More importantly, a four-base 3ʹ stick end produced by the homothallic switching endonuclease in cells could be recognized by telomerase, allowing the Tage system to effectively kill cancer cells in vivo. The Tage system could effectively and safely realize its in vivo application by using adeno-associated virus (AAV) as gene vector. The virus-loaded Tage system could significantly and specifically kill cancer cells in mice by intravenous drug administration without side effects or toxicity.
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Blackburn EH. Telomeres: do the ends justify the means? Cell. 1984;37:7–8.
Blackburn EH. Structure and function of telomeres. Nature. 1991;350:569–73.
Shampay J, Szostak JW, Blackburn EH. DNA sequences of telomeres maintained in yeast. Nature. 1984;310:154–7.
Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci USA. 1988;85:6622–6.
Williams TL, Levy DL, Maki-Yonekura S, Yonekura K, Blackburn EH. Characterization of the yeast telomere nucleoprotein core: Rap1 binds independently to each recognition site. J Biol Chem. 2010;285:35814–24.
Martinez P, Blasco MA. Role of shelterin in cancer and aging. Aging Cell. 2010;9:653–66.
Watson JD. Origin of concatemeric T7 DNA. Nat New Biol. 1972;239:197–201.
Wright WE, Shay JW. Historical claims and current interpretations of replicative aging. Nat Biotechnol. 2002;20:682–8.
Stewart SA, Bertuch AA. The role of telomeres and telomerase in cancer research. Cancer Res. 2010;70:7365–71.
Aschacher T, Wolf B, Enzmann F, Kienzl P, Messner B, Sampl S, et al. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines. Oncogene. 2016;35:94–104.
Deng Y, Chang S. Role of telomeres and telomerase in genomic instability, senescence and cancer. Lab Invest. 2007;87:1071–6.
Henson JD, Neumann AA, Yeager TR, Reddel RR. Alternative lengthening of telomeres in mammalian cells. Oncogene. 2002;21:598–610.
Colgin LM, Baran K, Baumann P, Cech TR, Reddel RR. Human POT1 facilitates telomere elongation by telomerase. Curr Biol. 2003;13:942–6.
Colgin LM, Reddel RR. Telomere maintenance mechanisms and cellular immortalization. Curr Opin Genet Dev. 1999;9:97–103.
Maciejowski J, de Lange T. Telomeres in cancer: tumour suppression and genome instability. Nat Rev Mol Cell Biol. 2017;18:175–86.
Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW. Telomerase activity in human germline and embryonic tissues and cells. Dev Genet. 1996;18:173–9.
Shay JW, Wright WE. Role of telomeres and telomerase in cancer. Semin Cancer Biol. 2011;21:349–53.
Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L, Garraway LA. Highly recurrent TERT promoter mutations in human melanoma. Science. 2013;339:957–9.
Jafri MA, Ansari SA, Alqahtani MH, Shay JW. Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies. Genome Med. 2016;8:69.
Mosoyan G, Kraus T, Ye F, Eng K, Crispino JD, Hoffman R, et al. Imetelstat, a telomerase inhibitor, differentially affects normal and malignant megakaryopoiesis. Leukemia. 2017;31:2458–67.
Tefferi A, Lasho TL, Begna KH, Patnaik MM, Zblewski DL, Finke CM, et al. A pilot study of the telomerase inhibitor imetelstat for myelofibrosis. N Engl J Med. 2015;373:908–19.
Khan FA, Pandupuspitasari NS, Chun-Jie H, Ao Z, Jamal M, Zohaib A, et al. CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases. Oncotarget. 2016;7:52541–52.
Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167–70.
Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471:602–7.
Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346:1258096.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.
Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol. 2014;32:1146–50.
Samanta MK, Dey A, Gayen S. CRISPR/Cas9: an advanced tool for editing plant genomes. Transgenic Res. 2016;25:561–73.
Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156:836–43.
Diede SJ, Gottschling DE. Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell. 1999;99:723–33.
Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994;266:2011–5.
Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10:459–66.
Sachdeva M, Sachdeva N, Pal M, Gupta N, Khan IA, Majumdar M, et al. CRISPR/Cas9: molecular tool for gene therapy to target genome and epigenome in the treatment of lung cancer. Cancer Gene Ther. 2015;22:509–17.
Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014;159:440–55.
Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014;514:380–4.
Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012;109:E2579–86.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. eLife. 2013;2:e00471.
Chavez A, Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan D, et al. Comparison of Cas9 activators in multiple species. Nat Methods. 2016;13:563–7.
Banerjee B, Sherwood RI. A CRISPR view of gene regulation. Curr Opin Syst Biol. 2017;1:1–8.
Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age. Science. 2018;359:1–10.
George LA. Hemophilia gene therapy comes of age. Blood Adv. 2017;1:2591–9.
Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med. 2017;377:1713–22.
Valdmanis PN, Kay MA. Future of rAAV gene therapy: platform for RNAi, gene editing, and beyond. Hum Gene Ther. 2017;28:361–72.
Nissim L, Wu MR, Pery E, Binder-Nissim A, Suzuki HI, Stupp D, et al. Synthetic RNA-based immunomodulatory gene circuits for cancer immunotherapy. Cell. 2017;171:1138–50 e15.
Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002;30:e47.
This work was supported by the National Natural Science Foundation of China (61571119) and the National Key Research and Development Program of China (2017YFA0205502).
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Dai, W., Xu, X., Wang, D. et al. Cancer therapy with a CRISPR-assisted telomerase-activating gene expression system. Oncogene 38, 4110–4124 (2019). https://doi.org/10.1038/s41388-019-0707-8
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