title: Zinc finger nucleases targeting the human papillomavirus E7 oncogene induce E7 disruption and a transformed phenotype in HPV16/18-positive cervical cancer cells.
abstract: OBJECTIVE
  Cervical cancer is mainly caused by infections of high-risk human papillomavirus (HR-HPV). Persistent expression of HR-HPV oncogenes E6 and E7 is implicated in malignant transformation. The aim was to provide proof-of-concept data to support use of zinc finger nucleases (ZFN) targeting HPV E7 to treat HPV-related cervical cancer.
  
  
  METHODS
  We designed and constructed ZFNs that could specifically recognize and cleave HPV16/18 E7 DNA. We tested the cleavage efficiency of selected ZFN16-E7-S2 and ZFN18-E7-S2 by using single-strand annealing (SSA) assay. Cell viability and colony formation assays were used to estimate the inhibition of cell growth that received treatments of ZFNs. Gene disruption of HPV E7 and downstream genes were examined by Western blotting. Cell apoptosis assay was used to test the specificity and efficiency of induction of HPV type-specific apoptosis. We also introduced xenograft formation assays to estimate the potential of inhibition of HPV-related disease.
  
  
  RESULTS
  We found ZFN16-E7-S2 and ZFN18-E7-S2 disrupted HPV E7 oncogenes in HPV16/18-positive cervical cancer cells. Both ZFNs effectively led to inhibition of type-specific cervical cancer cell growth, and specifically induced apoptosis of corresponding HPV16- and HPV18-positive cervical cancer cell lines. ZFN16-E7-S2 and ZFN18-E7-S2 also repressed xenograft formation in vivo.
  
  
  CONCLUSIONS
  ZFNs targeting HPV16/18 E7 could effectively induce disruption of E7 oncogenes and lead to type-specific and efficient growth inhibition and apoptosis of HPV-positive cells. ZFNs targeting HPV16/18 E7 oncogenes could be used as novel therapeutic agents for the treatment of HPV-related cervical cancer.
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Wencheng
authors.initials: W
authors.lastname: Ding
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Da
authors.initials: D
authors.lastname: Zhu
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Changlin
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Ji
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Kezhen
authors.initials: K
authors.lastname: Li
authors.affiliation: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Dan
authors.initials: D
authors.lastname: He
authors.affiliation: Center for Reproductive Medicine, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Xia
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. dma@tjh.tjmu.edu.cn huit71@sohu.com.
authors.forename: Ding
authors.initials: D
authors.lastname: Ma
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. dma@tjh.tjmu.edu.cn huit71@sohu.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D002472:Cell Transformation, Viral; D053960:DNA Breaks; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000235:genetics; D004195:Disease Models, Animal; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D055785:Gene Knockdown Techniques; D064593:Heterografts; D052162:Human papillomavirus 16 / Q000235:genetics*; D052161:Human papillomavirus 18 / Q000235:genetics*; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins / Q000235:genetics; D030361:Papillomavirus Infections / Q000150:complications*; D047368:Tumor Burden / Q000235:genetics; D002583:Uterine Cervical Neoplasms / Q000209:etiology* / Q000378:metabolism* / Q000473:pathology; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D004706:Endodeoxyribonucleases