TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy
Zheng Hu, … , Ding Ma, Hui Wang
Zheng Hu, … , Ding Ma, Hui Wang
Published December 15, 2014
Citation Information: J Clin Invest. 2015;125(1):425-436. https://doi.org/10.1172/JCI78206.
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Research Article Oncology

TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy

Abstract

Persistent HPV infection is recognized as the main etiologic factor for cervical cancer. HPV expresses the oncoproteins E6 and E7, both of which play key roles in maintaining viral infection and promoting carcinogenesis. While siRNA-mediated targeting of E6 and E7 transcripts temporarily induces apoptosis in HPV-positive cells, it does not eliminate viral DNA within the host genome, which can harbor escape mutants. Here, we demonstrated that specifically targeting E6 and E7 within host DNA with transcription activator–like effector nucleases (TALENs) induces apoptosis, inhibits growth, and reduces tumorigenicity in HPV-positive cell lines. TALEN treatment efficiently disrupted E6 and E7 oncogenes, leading to the restoration of host tumor suppressors p53 and retinoblastoma 1 (RB1), which are targeted by E6 and E7, respectively. In the K14-HPV16 transgenic mouse model of HPV-driven neoplasms, direct cervical application of HPV16-E7–targeted TALENs effectively mutated the E7 oncogene, reduced viral DNA load, and restored RB1 function and downstream targets transcription factor E2F1 and cycling-dependent kinase 2 (CDK2), thereby reversing the malignant phenotype. Together, the results from our study suggest that TALENs have potential as a therapeutic strategy for HPV infection and related cervical malignancy.

Authors

Zheng Hu, Wencheng Ding, Da Zhu, Lan Yu, Xiaohui Jiang, Xiaoli Wang, Changlin Zhang, Liming Wang, Teng Ji, Dan Liu, Dan He, Xi Xia, Tao Zhu, Juncheng Wei, Peng Wu, Changyu Wang, Ling Xi, Qinglei Gao, Gang Chen, Rong Liu, Kezhen Li, Shuang Li, Shixuan Wang, Jianfeng Zhou, Ding Ma, Hui Wang

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Figure 1

Screening of TALENs with different DNA-binding sites, TALEN architectures, and FokI variants.

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Screening of TALENs with different DNA-binding sites, TALEN architecture...
(A) Schematic presentation of the TALEN-mediated disruption of HPV oncogenes. The circular genome represents the episomal HPV genome, and the linear genome represents the integrated HPV genome. URR, upstream regulation region. (B) The locations of TALEN DNA-binding sites for HPV16 and HPV18 are indicated by arrows. The numbers in the names of TALENs indicate the first base of the TALEN spacer region. (C) Targeting efficiencies of the shown TALENs were measured using the SSA reporter assay. The fold inductions of the TALENs relative to the controls are shown at the top of the columns. RLU, relative luminescence unit. Data represent mean ± SD; n = 3 per group. (D) Schematic diagram of the initial +231 T512 and the +63 truncated T512 architectures and FokI variants. (Detailed sequences of FokI variants can be found in Supplemental Note 1.) (E) The corresponding nuclease activities of these combinations, as measured using the SSA reporter assay. The fold inductions of the TALENs relative to the control, Luci-512, are shown on the right of the columns. The ZFN GZF1/3 was used as a positive control, and the fold induction was related to that of pSSA rep3-1. RVDs, repeat variable diresidues. Data represent mean ± SD; n = 3 per group. (F) The toxicity profiles of the combinations shown were assessed using the SSA reporter assay. Renilla luminescence signals were constitutively high in the absence of TALEN/ZFN toxicity. Data represent mean ± SD; n = 3 per group. All experiments were performed in triplicate.