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J. Virol., 02 1997, 1397-1404, Vol 71, No. 2
Copyright © 1997, American Society for Microbiology

High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells

DS An, Y Koyanagi, JQ Zhao, R Akkina, G Bristol, N Yamamoto, JA Zack and IS Chen
Department of Microbiology & Immunology, UCLA School of Medicine, Los Angeles, California 90095-1678, USA.

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy- 1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor- derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T- lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.

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