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  • Viral Transfer Technology
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Adenoviral vector design for high-level transgene expression in primitive human hematopoietic progenitors

Gene Therapy volume 7, pages 2132–2138 (2000)Cite this article

Abstract

Adenoviral vector-mediated transient gene expression can provide new possibilities for ex vivo manipulation of quiescent hematopoietic stem cells (HSC). In order to define a suitable expression cassette for high levels of transgene expression in HSCs, we have studied the level of transgene expression in human CD34+CD38− cells using adenoviral vectors with various gene expression cassettes encoding the enhanced green fluorescence protein (EGFP) gene. CD34+ hematopoietic cells were cultured in serum-free medium with megakaryocyte growth and development factor (MGDF) alone for supporting the survival of primitive progenitors or with MGDF, c-kit ligand (KL) and flt3 ligand (FL) for inducing proliferation of primitive progenitors. With all the vectors tested, higher percentages of EGFP expressing cells were found in CD34+CD38− cells than those in CD34+CD38high cells from all donors tested. The phosphoglycerate kinase (PGK)-1 promoter was found to allow higher levels of EGFP expression than the human cytomegalovirus (HCMV) promoter in CD34+CD38− cells. Replacing the SV40 polyadenylation signal with the human β-globin gene IVS2 and polyadenylation signal in the expression cassette (Ad5xPGK-EGFP-β-globin) enhanced the level of EGFP expression markedly further. These results provide a guideline for the development of adenoviral vectors for gene expression in human primitive hematopoietic progenitor cells.

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Acknowledgements

These studies were generously supported by the Medical Faculty, University of Lund, the Swedish Cancer Society and The Medical Research Council, Sweden by grants to Stefan Karlsson. The authors gratefully acknowledge Dr Johan Richter and other colleagues at the Department of Hematology (Lund University Hospital) for performing the human bone marrow aspirations and thank our bone marrow volunteers for their donations. We are indebted to Dr Saemundur Gudmundsson and the staff of The Department of Obstetrics and Gynecology, Malmö for collecting umbilical cord blood samples. We thank Drs Ian K McNiece, Janet Nichol (Amgen, Thousand Oaks, CA) for generously supplying MGDF and KL, and Dr Stewart Lyman (Immunex, Seattle, WA) for providing FL for these studies.

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  1. Department of Molecular Medicine and Gene Therapy, Lund University, Sweden

    X Fan, A Brun & S Karlsson

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Fan, X., Brun, A. & Karlsson, S. Adenoviral vector design for high-level transgene expression in primitive human hematopoietic progenitors. Gene Ther 7, 2132–2138 (2000). https://doi.org/10.1038/sj.gt.3301352

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