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Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

Gene Therapy volume 12, pages 1424–1433 (2005)Cite this article

Abstract

Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1α promoter (EF1α promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/β-actin promoter with β-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+CD38low/− and CD34+AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

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Acknowledgements

We thank Tomomi Sasaki and Takashi Fukushima for technical assistance. We would also like to thank Dr J Miyazaki and Dr RG Hawley for kindly providing the CA promoter and the MSCV promoter, respectively. This work was supported in part by a Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by grants for Health and Labour Sciences Research from the Ministry of Health, Labour, and Welfare of Japan.

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Authors and Affiliations

  1. Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Osaka, Japan

    F Sakurai, K Kawabata & H Mizuguchi

  2. Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, Tokyo, Japan

    T Yamaguchi

  3. Pharmaceuticals and Medical Devices Agency, Tokyo, Japan

    T Hayakawa

  4. Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan

    H Mizuguchi

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Sakurai, F., Kawabata, K., Yamaguchi, T. et al. Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities. Gene Ther 12, 1424–1433 (2005). https://doi.org/10.1038/sj.gt.3302562

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