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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 445-452
High levels of lymphoid expression of enhanced green fluorescent
protein in nonhuman primates transplanted with cytokine-mobilized
peripheral blood CD34+ cells
Robert E. Donahue,
Robert P. Wersto,
James A. Allay,
Brian A. Agricola,
Mark E. Metzger,
Arthur W. Nienhuis,
Derek A. Persons, and
Brian P. Sorrentino
From the Hematology Branch, National Heart, Lung, and Blood
Institute, Rockville, MD, and Experimental Hematology, St. Jude
Children's Research Hospital, Memphis, TN.
We have used a murine retrovirus vector containing an enhanced green
fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow
vector-expressing cells in the peripheral blood (PB) of transplanted
rhesus macaques. Cytokine mobilized CD34+ cells were
transduced with an amphotropic vector that expressed EGFP and a
dihydrofolate reductase cDNA under control of the murine stem
cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high
concentrations of the flt-3 ligand and stem cell
factor. Following transplantation of the transduced cells, up to 55%
EGFP-expressing granulocytes were obtained in the peripheral
circulation during the early posttransplant period. This level of
myeloid marking, however, decreased to 0.1% or lower within 2 weeks.
In contrast, EGFP expression in PB lymphocytes rose from 2%-5%
shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and
Southern blot analysis, and EGFP expression was observed in CD4+, CD8+, CD20+, and
CD16/56+ lymphocyte subsets. EGFP expression was only
transiently detected in red blood cells and platelets soon after
transplantation. Such sustained levels of lymphocyte marking may be
therapeutic in a number of human gene therapy applications that require
targeting of the lymphoid compartment. The transient appearance of
EGFP+ myeloid cells suggests that
transduction of a lineage-restricted myeloid progenitor capable of
short-term engraftment was obtained with this protocol.
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