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Journal of Integrative Medicine ›› 2021, Vol. 19 ›› Issue (6): 515-525.doi: 10.1016/j.joim.2021.09.004

• Original Experimental Research • Previous Articles     Next Articles

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Gai Rana,b, Xi-lin Fenga, Yi-lin Xiea, Qing-yun Zhenga, Peng-peng Guoc, Ming Yanga, Ying-lu Fengc, Chen Linga,b, Li-qing Zhud, Chen Zhonga   

  1. a. State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China
    b. Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 32611, USA
    c. Department of Traditional Chinese Medicine, Chinese People’s Liberation Army 971 Hospital, Qingdao 266071, Shandong Province, China
    d. Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
  • Received:2020-04-03 Accepted:2021-05-10 Online:2021-11-12 Published:2021-12-21
  • Contact: Chen Zhong E-mail:zhongchen@fudan.edu.cn

Objective

Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.

Methods

A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.

Results

The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.

Conclusion

HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Key words: Trichosanthin, miR122 target sequence, Recombinant adeno-associated virus vector, Gene therapy, Liver cancer

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