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

被引:14
作者
Gatlin, J
Douglas, J
Evans, JT
Collins, RH
Wendel, GD
Garcia, V
机构
[1] Univ Texas, SW Med Ctr, Dept Internal Med, Div Infect Dis Y9 206, Dallas, TX 75235 USA
[2] SyStem Inc, Palo Alto, CA 94304 USA
[3] Univ Texas, SW Med Ctr, Dept Obstet & Gynecol, Dallas, TX 75235 USA
关键词
D O I
10.1089/10430340050129558
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 (DHFRL22Y) For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFRL22Y 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 DHFRL22Y+ 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.
引用
收藏
页码:1949 / 1957
页数:9
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