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Radio‐activated gene therapy has been developed as a novel therapeutic strategy against cancer; however, expression of therapeutic gene in peritumoral tissues will result in unacceptable toxicity to normal cells. To restrict gene expression in targeted tumor mass, we used hypoxia and radiation tolerance features of tumor cells to develop a synthetic  AND  gate genetic circuit through connecting radiation sensitivity promoter  cArG  6 , heat shock response elements  SNF 1,  HSF 1 and  HSE  4  with retroviral vector plxsn. Their construction and dynamic activity process were identified through downstream enhanced green fluorescent protein and wtp53 expression in non‐small cell lung cancer A549 cells and in a nude mice model. The result showed that  AND  gate genetic circuit could be activated by lower required radiation dose (6 Gy) and after activated,  AND  gate could induce significant apoptosis effects and growth inhibition of cancer cells  in vitro  and  in vivo . The radiation‐ and hypoxia‐activated  AND  gate genetic circuit, which could lead to more powerful target tumoricidal activity represented a promising strategy for both targeted and effective gene therapy of human lung adenocarcinoma and low dose activation character of the  AND  gate genetic circuit implied that this model could be further exploited to decrease side‐effects of clinical radiation therapy.

p53 activated by AND gate genetic circuit under radiation and hypoxia for targeted cancer gene therapy