High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells

We have used a murine retrovirus vector containing an enhanced green fluore scent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-ex pressing cells in the peripheral blood (PB) of transplanted rhesus macaques , Cytokine:mobilized CD34(+) cells were transduced with an amphotropic vect or 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 concentratio ns of the flt-3 ligand and stem cell factor. Following transplantation of t he transduced cells, up to 55% EGFP-expressing granulocytes were obtained i n the peripheral circulation during the early posttransplant period. This l evel 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 fo llowing transplantation to 10% or greater by week 5, After 10 weeks, the le vel of expression in PB lymphocytes continued to remain at 3%-5% as measure d by both flow cytometry and Southern blot analysis, and EGFP expression wa s observed in CD4(+), CD8(+), CD20(+), and CD15/56(+) lymphocyte sub-sets. EGFP expression was only transiently detected in red blood cells and platel ets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that req uire targeting of the lymphoid compartment, The transient appearance of EGF P(+) myeloid cells suggests that transduction of a lineage-restricted myelo id progenitor capable of short-term engraftment was obtained with this prot ocol. (Blood, 2000; 95:445-452) (C) 2000 by The American Society of Hematol ogy.