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  • Nonviral Transfer Technology
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High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery

Gene Therapy volume 7, pages 1431–1437 (2000)Cite this article

Abstract

The design of effective gene delivery systems for gene transfer in primary human blood cells is important both for fundamental hematopoiesis research and for cancer gene therapy strategies. Here, we evaluated electroporation as a nonviral means for transfection of activated human T lymphocytes and adult bone marrow (BM) CD34+ cells. We describe optimal culture and electroporation parameters for efficient gene delivery in prestimulated T lymphocytes (16.3 ± 1.3%), as well as 2-day cultured adult BM CD34+ cells (29.6 ± 4.6%). PHA-stimulated T cells were most receptive for transfection after 48h of in vitro culture, while T cells stimulated by CD3 cross-linking and interleukin (IL)-2 achieved maximum transfection levels after 72 h of prestimulation. Kinetic analysis of EGFP expression revealed that activated T lymphocytes maintained transgene expression at high levels for a prolonged period. In addition, fresh unstimulated BM CD34+ cells were consistently transfected (5.2 ± 0.4%) with minimal cytotoxicity (<5%), even without preliminary cd34+ cell purification. Both T cells and CD34+ cells retained their phenotype and functional capacity after electroporation. These results demonstrate that electroporation is a suitable nonviral transfection technique that may serve applications in gene therapy protocols using T lymphocytes or CD34+ cells.

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Acknowledgements

This work was supported by grant No. 3.0109.96 and No. G.0157.99 of the Fund for Scientific Research, Flanders, Belgium (FWO), by a grant of the Scientific Committee of the ASLK-financed Cancer Research and by gifts of Mrs Ellie Van Belleghem to the HEBA Foundation (Foundation for Hematology, Bone Marrow Transplantation and Blood Transfusion, Antwerp) in memory of her late mother Louisa De Saedeleer. We would like to thank Dr SC Lyman and Dr EK Thomas (Immunex Corporation, Seattle, WA, USA) for providing us with Flt-3 ligand, Dr John Wunderlich (NIH, Bethesda, USA) for the 1235 Melan-A-specific TIL clone. VVT is a research assistant of the FWO. RW is a holder of a research grant of the Vlaamse Kankerliga. PP is holder of a research grant of the Institute for Science and Technology (IWT).

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Authors and Affiliations

  1. Laboratory of Experimental Hematology, Antwerp University Hospital (UIA/UZA), Belgium

    V F I Van Tendeloo, R Willems, P Ponsaerts, M Lenjou, G Nijs, DR Van Bockstaele & ZN Berneman

  2. Department of Molecular Genetics, Laboratory of Molecular Genetics, University of Antwerp (UIA), Flanders Interuniversity Institute for Biotechnology (VIB), Belgium

    V F I Van Tendeloo & C Van Broeckhoven

  3. Department of Cardiac Surgery, Middelheim General Hospital, Antwerp, Belgium

    M Vanhove, P Muylaert & P Van Cauwelaert

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  1. V F I Van Tendeloo
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Van Tendeloo, V., Willems, R., Ponsaerts, P. et al. High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery. Gene Ther 7, 1431–1437 (2000). https://doi.org/10.1038/sj.gt.3301252

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