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TALEN based HPV-E7 editing triggers necrotic cell death in cervical cancer cells
Sumitra Shankar1, Deepti Prasad1, Rahul Sanawar1
1Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thycaud. P. O., Thiruvananthapuram-14, Kerala, India.
Abstract:
Human Papillomavirus E7 and E6 oncoproteins have been considered as suitable candidate anti-viral targets since they cause malignant conversion in cervical cancers. Transcription Activator-Like Effector Nucleases (TALENs) are recent editing tools to knockout genes by inducing double stranded breaks at specific sites in the genome. In here, we have designed specific TALENs to target E7 and analyzed their efficiency in inducing cell death in cervical cancer cells. We found that designed TALENs could yield about 10-12% editing activity as observed from T7E1 and nuclease resistance assays. Down-regulation of E7 and E6 was further evident at the transcript as well as proteins levels indicating that the selected TALENs were effective. TALEN-mediated E7 editing led to cell death as ascertained by cell cycle and Annexin V assays. Annexin profiling suggested that cell death could be due to necrosis as observed by upregulation of necrotic markers such as LDH A, Rip-1, and Cyclophilin A. Necrosis appears to be a better therapeutic response as it could further activate pro-inflammatory cytokines to attract immune cells to eliminate HPV-integrated cells and therefore TALEN editing strategy has the potential to be a promising tool as an adjuvant therapy in cervical cancer along with surgery.
Insights
Transcription Activator-Like Effector Nucleases (TALENs) effectively targeted Human Papillomavirus E7 in cervical cancer cells, inducing cell death. This gene-editing approach shows promise as an adjuvant therapy for cervical cancer.
Area of Science:
- Oncology
- Molecular Biology
- Gene Editing
Background:
- Human Papillomavirus (HPV) oncoproteins E6 and E7 drive cervical cancer progression.
- Targeting these oncoproteins presents a viable anti-viral strategy.
Purpose of the Study:
- To design and assess Transcription Activator-Like Effector Nucleases (TALENs) targeting the HPV E7 oncoprotein.
- To evaluate the efficacy of TALENs in inducing cell death in cervical cancer cells.
Main Methods:
- Designed TALENs specific for the HPV E7 gene.
- Assessed TALEN editing efficiency using T7E1 and nuclease resistance assays.
- Analyzed E7/E6 down-regulation at transcript and protein levels.
- Evaluated TALEN-induced cell death via cell cycle and Annexin V assays.
- Investigated cell death mechanisms, including necrosis markers.
Main Results:
- TALENs achieved 10-12% editing activity against the E7 gene.
- Significant down-regulation of E7 and E6 oncoproteins was observed.
- TALEN editing induced cervical cancer cell death, primarily through necrosis.
- Necrotic markers like LDH A, Rip-1, and Cyclophilin A were upregulated.
Conclusions:
- TALENs are effective in editing HPV E7 and inducing cancer cell death.
- Necrosis as a cell death mechanism may enhance immune response against HPV-infected cells.
- TALEN-based gene editing holds potential as an adjuvant therapy for cervical cancer.
