TALEN based HPV-E7 editing triggers necrotic cell death in cervical cancer cells

Shankar, Sumitra; Prasad, D. D. K.; Sanawar, Rahul; Das, Ani V.; Pillai, M. Radhakrishna

doi:10.1038/s41598-017-05696-0

Cited by 35 publications

(23 citation statements)

References 41 publications

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“…6 b). We recently reported that TALENs successfully edit E7 gene in SiHa cells [ 20 ], leading to necrotic cell death. This was further corroborated by RT-PCR analysis for the transcripts corresponding to E7 in both SiHa and CaSki cells.…”

Section: Resultsmentioning

confidence: 99%

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Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

et al. 2018

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BackgroundGene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome.MethodsHPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole.ResultsAlthough ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity.ConclusionsOur study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.Electronic supplementary materialThe online version of this article (10.1186/s12935-018-0666-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

“…Since no useful target sites were obtained for ZFNs, we designed TALENs targeting E6 which also did not yield any editing activity in these cell lines. We recently showed that TALENs successfully edited E7 gene in SiHa cells [ 20 ]. We extended this editing study to another HPV +ve cell line CaSki and found similar editing efficiency.…”

Section: Introductionmentioning

confidence: 99%

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

et al. 2018

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“…HPV E7 was found to have no such effect; instead, it showed minimal pro-apoptotic activity, which was nullified by the strongly anti-apoptotic consequences of E6 activity (Aguilar-Lemarroy et al, 2002). In addition to the apoptotic modulation, a noteworthy observation was made by Shankar et al (2017), wherein TALEN-based editing of HPV E7 was found to induce necrosis in cervical cancer cells, which has been proved to hold better therapeutic response.…”

Section: Resisting Cell Deathmentioning

confidence: 99%

Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy

2020

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“…Most recently, genome editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 protein (CRISPR/Cas9) in the treatment of HPV-induced cancers have been shown to induce apoptosis, inhibit growth and reduce tumorigenicity in HPV+ cell lines, and to reduce the viral load in transgenic mouse models [118] , [119] , [120] , [121] , [122] , [123] , [124] . A phase I study to determine the efficacy of ZFN-603 and ZFN-758, which can cleave HPV-16/18 E7, in the treatment of cervical precancerous lesions has recently been completed ( NCT02800369 ).…”

Section: Therapeutic Vaccine Strategiesmentioning

confidence: 99%

Therapeutic vaccines for high-risk HPV-associated diseases

Chabeda

Yanez

Lamprecht

et al. 2018

Papillomavirus Research

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Cancer is the second leading cause of death worldwide, and it is estimated that Human papillomavirus (HPV) related cancers account for 5% of all human cancers. Current HPV vaccines are extremely effective at preventing infection and neoplastic disease; however, they are prophylactic and do not clear established infections. Therapeutic vaccines which trigger cell-mediated immune responses for the treatment of established infections and malignancies are therefore required. The E6 and E7 early genes are ideal targets for vaccine therapy due to their role in disruption of the cell cycle and their constitutive expression in premalignant and malignant tissues. Several strategies have been investigated for the development of therapeutic vaccines, including live-vector, nucleic acid, peptide, protein-based and cell-based vaccines as well as combinatorial approaches, with several vaccine candidates progressing to clinical trials. With the current understanding of the HPV life cycle, molecular mechanisms of infection, carcinogenesis, tumour biology, the tumour microenvironment and immune response mechanisms, an approved HPV therapeutic vaccine seems to be a goal not far from being achieved. In this article, the status of therapeutic HPV vaccines in clinical trials are reviewed, and the potential for plant-based vaccine production platforms described.

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TALEN based HPV-E7 editing triggers necrotic cell death in cervical cancer cells

Cited by 35 publications

References 41 publications

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy

Therapeutic vaccines for high-risk HPV-associated diseases

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