Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia

Wayengera, Misaki

doi:10.1186/1742-4682-9-30

Cited by 8 publications

(7 citation statements)

References 48 publications

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“…Since E6 gene had few GNN triplets it indicated that ZiFiT could not provide suitable hits. On similar lines, in a study by Wayengara, where zinc finger arrays were used to target HPV 16 and HPV 18 genome using ZiFiT software, only a set of unpaired zinc finger arrays of length 9 bp were obtained for E6 gene [ 32 ]. It is known that zinc fingers that recognize GNN triplets are well characterized and have been used to successfully design ZFNs to knockout several genes.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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

et al. 2018

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“…Similar results in another study showed that using ZFNs to target HPV E7 induced specific shear of the E7 gene and attenuated its malignant biological effect. 150 Wayengera et al 151 computationally generated paired zinc-finger arrays (pZFAs) to target and cleave the genomic DNA of HPV-type 16 and 18, respectively. The authors highlighted the therapeutic effect of ZFN-mediated gene disruption in HPV 16/18, which was achieved when HPV-derived viral plasmids or vectors were introduced into precancerous lesions to realize targeted mutagenesis and gene-therapeutic reversal of cervical neoplasia.…”

Section: Viral Diseasesmentioning

confidence: 99%

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

Li¹,

Yang²,

Hong³

et al. 2020

Sig Transduct Target Ther

1,567

869

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Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

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“…The similar domains of E6 and E7 proteins allow the formation of telomeric complexes that possess interesting biological activities (78). The mutations in the zinc finger domain of E7 do not affect the ability to bind and deteriorate the pRB, but they do abrogate its ability to immortalize cells (79). In the human papillomavirus 83 (HPV83m) that produced cervical cancer, five mutations were found in the second zinc finger domain of E6, an important domain for protein-protein or protein-DNA interactions.…”

Section: Hpv E6/e7 Proteins and Zinc Fingersmentioning

confidence: 99%

Relevance of infection with human papillomavirus: The role of the p53 tumor suppressor protein and E6/E7 zinc finger proteins

Ruttkay-Nedecký

Jiménez

Nejdl

et al. 2013

International Journal of Oncology

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Human papillomaviruses (HPV) are small circular, double-stranded DNA viruses infecting epithelial tissues. HPV types can be classified both as high-risk or low-risk. Of the more than 120 different identified types of HPV, the majority are involved in infections of the genital tract, cancer of the cervix, vulva, vagina and penis, and of non-anogenital localizations, such as the head and neck areas. From the point of view of the infection, human papillomaviruses have developed several molecular mechanisms to enable infected cells to suppress apoptosis. This review provides a comprehensive and critical summary of the current literature that focuses on cervical carcinoma and cancer of the head and neck caused by HPV. In particular, we discuss HPV virology, the molecular mechanisms of carcinogenesis, the role of the tumor suppressor protein p53 and the E6/E7 zinc finger proteins. Classification of HPV according to diagnosis is also described.

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Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia

Cited by 8 publications

References 48 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

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

Relevance of infection with human papillomavirus: The role of the p53 tumor suppressor protein and E6/E7 zinc finger proteins

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