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An efficient construction of conditionally replicating adenoviruses that target tumor cells with multiple factors

Gene Therapy volume 12, pages 1385–1393 (2005)Cite this article

Abstract

Despite the enormous potential of conditionally replicating adenoviruses (CRAs), the time-consuming and laborious methods required to construct CRAs have hampered both the development of CRAs that can specifically target tumors with multiple factors (m-CRA) and the efficient analysis of diverse candidate CRAs. Here, we present a novel method for efficiently constructing diverse m-CRAs. Elements involving viral replication, therapeutic genes, and adenoviral backbones were separately introduced into three plasmids of P1, P2, and P3, respectively, which comprised different antibiotic resistant genes, different ori, and a single loxP (H) sequence. Independently constructed plasmids were combined at 100% accuracy by transformation with originally prepared Cre and specific antibiotics in specific Escherichia coli; transfection of the resulting P1+2+3 plasmids into 293 cells efficiently generated m-CRAs. Moreover, the simultaneous generation of diverse m-CRAs was achieved at 100% accuracy by handling diverse types of P1+2 and P3. Alternatively, co-transfection of P1+3 and P2 plasmids into Cre-expressing 293 cells directly generated m-CRA with therapeutic genes. Thus, our three-plasmid system, which allows unrestricted construction and efficient fusion of individual elements, should expedite the process of generating, modifying, and testing diverse m-CRAs for the development of the ideal m-CRA for tumor therapy.

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Acknowledgements

This study was supported in part by a grant for Cooperation of Innovative Technology and Advanced Research in Evolutional Area (CITY AREA) and a Grant-in-Aid for Scientific Research on Priority Areas (C) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, a grant from the Uehara Memorial Foundation and Health and Labour Sciences Research Grants for Third Term Comprehensive Control Research for Cancer from the Ministry of Health, Labour and Welfare, Japan. We thank Izumu Saito, Jun-ichi Miyazaki, Howard Fine, Satoru Kyo, Z Sheng Guo, and Mark A Kay for providing materials and David Cochran for editing the manuscript.

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Authors and Affiliations

  1. Division of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute of Brain Diseases, Kurume University, Kurume, Japan

    S Nagano & K Kosai

  2. Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

    S Nagano & S Komiya

  3. Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan

    S Nagano & K Kosai

  4. Department of Gene Therapy and Regenerative Medicine, Gifu University School of Medicine, Gifu, Japan

    H Oshika & K Kosai

  5. Department of Cardiology, Respiratory and Nephrology, Regeneration & Advanced Medical Science, Graduate School of Medicine, Gifu University, Gifu, Japan

    H Fujiwara

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Nagano, S., Oshika, H., Fujiwara, H. et al. An efficient construction of conditionally replicating adenoviruses that target tumor cells with multiple factors. Gene Ther 12, 1385–1393 (2005). https://doi.org/10.1038/sj.gt.3302540

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