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Published on: January 5, 2018
Cancer gene therapy by NF-κB-activated cancer cell-specific expression of CRISPR/Cas9 targeting telomeres
Wei Dai1, Jian Wu1, Danyang Wang1
1State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China.
Abstract:
The transcription factor NF-κB is an attractive target for cancer therapy due to its over-activation in all tumours; however, NF-κB inhibitors developed in the past decades rarely became drugs due to undesirable side effects. In this study, we developed a gene therapy strategy named NF-κB-activated gene expression (Nage), which could induce the death of cancer cells in vitro and in vivo by utilising NF-κB over-activity in cancer cells, but had no effects on normal cells. Nage was consisted of an NF-κB-specific promoter formed by fusing an NF-κB decoy sequence with a minimal promoter, which could be bound by the intracellular over-activated NF-κB and thus activated the expression of downstream effector genes in an NF-κB-specific manner. In this study, we first confirmed the cancer cell-specific over-activation of NF-κB and then tested the cancer cell specificity of the Nage vector by expressing the reporter gene ZsGreen in various in vitro cultivated cells. We next demonstrated that the Nage vector could be used to express CRISPR/Cas9 protein only in cancer cells. The Cas9 protein was then guided by a sgRNA targeting telomeric DNA and induced cancer cell death. The Nage vector expressing Cas9/sgRNA could be packaged into adeno-associated virus (AAV) and intravenously injected to inhibit tumour growth in mice without visible side effects and toxicity.
Insights
A novel gene therapy strategy, NF-κB-activated gene expression (Nage), targets cancer cells by exploiting their overactive NF-κB pathway. This approach induces cancer cell death while sparing normal cells, offering a safer therapeutic alternative.
Area of Science:
- Oncology
- Gene Therapy
- Molecular Biology
Background:
- Nuclear factor kappa B (NF-κB) is constitutively active in many cancers, making it a promising therapeutic target.
- Previous NF-κB inhibitors have faced challenges due to off-target effects and toxicity.
Purpose of the Study:
- To develop a cancer-specific gene therapy strategy utilizing the overactive NF-κB pathway.
- To engineer a system that selectively induces cancer cell death with minimal impact on normal tissues.
Main Methods:
- Designed an NF-κB-activated gene expression (Nage) vector with an NF-κB-specific promoter.
- Validated cancer cell-specific NF-κB activity and Nage vector targeting in vitro.
- Utilized Nage to express CRISPR/Cas9 targeting telomeric DNA in cancer cells.
- Delivered the Nage-CRISPR/Cas9 system via adeno-associated virus (AAV) in vivo.
Main Results:
- Confirmed NF-κB over-activation is specific to cancer cells.
- Nage vector demonstrated cancer cell-specific reporter gene expression.
- Nage-mediated CRISPR/Cas9 expression successfully induced cancer cell death.
- AAV-delivered Nage vector inhibited tumor growth in mice without observable toxicity.
Conclusions:
- The Nage gene therapy strategy offers a novel, cancer-specific approach to cancer treatment.
- This method leverages tumor-specific NF-κB activity for targeted cancer cell ablation.
- Nage-based gene therapy shows potential as a safe and effective cancer treatment modality.
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