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Differentiation of Trev-Transduced CD34+ Progenitors into Cells Coexpressing CD4 and CCR5 G. ALTER*, N. F. BERNARD, D. COURNOYER, and C. M. TSOUKAS.
Immune Deficiency Treatment Ctr. and McGill Univ. Hlth. Ctr., Montreal Gen. Hosp., Canada Background: The Trev retroviral vector can downregulate the expression of the tat- and rev- dependent gene expression and increase survival in HIV infected cells.
Objectives: To determine whether Trev-transcduced CD34+ cells can differentiate into cells co-expressing CD4 and CCR5.
Methods: CD34+ cells were enriched from the mononuclear white blood cell population collected by apheresis from individuals undergoing G-CSF mobilization of "stem" cells. CD34+ cells were transduced with either Trev or a control vector. Transduction efficiency was determined by picking individual colonies originating from transduced cells and screening them for the expression of transfected DNA by nested PCR amplification, gel electrophoresis and visualization on an ethidium bromide stained gel. In separate experiments, transduced CD34+ cells were cultured on AFT024 bone marrow stroma in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor for up to 6 weeks. Each week the cellular composition of cultured cells was monitored for expression of CD4 and CCR5 and other cell surface markers by FACS analysis.
Results: Transduction efficiency was 90%. Cells expressing CD4 and CCR5 began to appear at 4 weeks of culture and increased in percentage at weeks 5 and 6. CD4+/CCR5+ cell percentages remained below 5%.
Conclusions: High efficiency gene transfer of Trev can be obtained in clonogenic human hematopoietic cells. These CD34+ cells can differentiate into cells potentially susceptible to HIV infection by co-expression of CD4 and CCR5. The next phase of these experiments will be to challenge cells with HIV isolates and determine whether Trev transduced cells are more resistant to HIV than those transduced with a control vector.
Key Words: genetic transduction, stem cells, tat and rev
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