In vitro selection of lentivirus vector-transduced human CD34+ cells

J. Gatlin, J. Douglas, J. T. Evans, R. H. Collins, G. D. Wendel, J. V. Garcia

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14 Scopus citations

Abstract

Human CD34+ cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous hematopoietic disorders. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human hematopoietic cells, we investigated a dominant selection strategy using a variant of the DHFR gene (DHFR(L22Y)). For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFR(L22Y). Here we demonstrate efficient in vitro selection and enrichment of lentivirus vector-transduced human CD34+ hematopoietic cells from fetal liver, umbilical cord blood, bone marrow, and peripheral blood after cytokine mobilization. Growth of transduced human CD34+ cells in semisolid culture under selective pressure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14). Selection for DHFR(L22Y)+ cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting that development of such strategies holds promise for future in vivo application.

Original languageEnglish (US)
Pages (from-to)1949-1957
Number of pages9
JournalHuman Gene Therapy
Volume11
Issue number13
DOIs
StatePublished - Sep 1 2000

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ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Gatlin, J., Douglas, J., Evans, J. T., Collins, R. H., Wendel, G. D., & Garcia, J. V. (2000). In vitro selection of lentivirus vector-transduced human CD34+ cells. Human Gene Therapy, 11(13), 1949-1957. https://doi.org/10.1089/10430340050129558