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Cancer gene therapy by NF-κB-activated cancer cell-specific expression of CRISPR/Cas9 targeting telomeres

Gene Therapy volume 27, pages 266–280 (2020)Cite this article

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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.

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Fig. 1: Relative NF-κB RelA protein abundance (i.e. NF-κB activity) in various cell lines.
Fig. 2: Expression of ZsGreen with the Nage vector in various cell lines.
Fig. 3: Expression of Cas9-TsgRNA with the Nage vector in various cell lines.
Fig. 4: Quantitative telomere length measurement with qPCR.
Fig. 5: Evaluation of rAAV.
Fig. 6: Gene therapy of xenografted cancers in mice with the recombinant AAV packaged with the Nage vector expressing Cas9 and TsgRNA (rAAV-TsgRNA-DMP-Cas9).

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Funding

This work was supported by the National Natural Science Foundation of China (61971122) and the National Key Research and Development Programme of China (2017YFA0205502).

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  1. State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China

    Wei Dai, Jian Wu, Danyang Wang & Jinke Wang

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  1. Wei Dai
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JKW conceived the study and designed experiments. WD designed and performed main experiments. JW and DYW prepared reagents and performed partial experiments. JKW and WD wrote the paper with support from all authors.

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Correspondence to Jinke Wang.

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Dai, W., Wu, J., Wang, D. et al. Cancer gene therapy by NF-κB-activated cancer cell-specific expression of CRISPR/Cas9 targeting telomeres. Gene Ther 27, 266–280 (2020). https://doi.org/10.1038/s41434-020-0128-x

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