Effects of busulfan dose escalation on engraftment of infant rhesus monkey hematopoietic stem cells after gene marking by a lentiviral vector.

OBJECTIVE: Non-myeloablative cytoreduction is used in clinical hematopoietic stem cell gene therapy trials to increase engraftment of gene-modified cells. We utilized an infant rhesus monkey model to identify an optimal dosage of busulfan that results in efficient long-term gene marking with minimal toxicities. METHODS: Bone marrow (BM) was harvested, followed by a single 2-hour intravenous infusion of busulfan at escalating dosages of 0 to 160 mg/m(2). CD34(+) cells were immunoselected from BM, transduced overnight with a simian immunodeficiency virus-based lentiviral vector carrying a non-expressed marker gene, and injected intravenously 48 hours post-busulfan administration. Pharmacokinetics were assessed, as well as adverse effects and peripheral blood and BM gene marking. RESULTS: Increasing dosages of busulfan resulted in increased area-under-the-curve (AUC) with some variability at each dosage level, suggesting interindividual variation in clearance. Blood chemistries were normal and no adverse effects were observed as a result of busulfan infusion. At 120 and 160 mg/m(2), transient neutropenia and thrombocytopenia were noted but not lymphopenia. Over the 6 months of study posttransplantation, a busulfan dosage-related increase in gene marking was observed ranging from undetectable (no busulfan) up to 0.1% gene-containing cells in animals achieving the highest busulfan AUC. This corresponds to a more than 100-fold increase in gene marking over the busulfan dosage range studied. CONCLUSIONS: These data indicate that increased gene marking of hematopoietic stem cells can be achieved by escalating busulfan dosages from 40 to 160 mg/m(2) without significant toxicity in infant nonhuman primates.

Effect of age on the frequency, cell cycle, and lineage maturation of rhesus monkey (Macaca mulatta) CD34+ and hematopoietic progenitor cells.

The effects of maturation and aging on hematopoietic progenitor cells, blood and bone marrow from second- and third-trimester fetal, newborn, infant, adult, and aged rhesus monkeys (Macaca mulatta) were analyzed. CD34(+) cells were immunoselected and stained with propidium iodide for cell cycle analysis. Blood and bone marrow mononuclear cells were plated in methylcellulose, and erythroid and myeloid progenitors were grown and counted. A higher frequency of circulating CD34(+)CD38(-) and CD34(+)DR(-) cells was observed in second-trimester fetuses compared with the other age groups. The frequency of bone marrow CD34(+)CD38(-) and CD34(+)DR(-) cells declined in adult and aged animals when compared with the younger age groups. Cell-cycle analysis showed 4.5% second-trimester fetal bone marrow CD34(+) cells entering the G(2)/M phase, compared with 1.7% CD34(+) cells in aged animals. More than 95% of circulating CD34(+) cells remained quiescent for most age groups, except for second-trimester fetuses. Adult marrow myeloid progenitors were found in a lower quantity when compared with third-trimester fetuses, whereas erythroid progenitors were greatest in early-gestation fetuses and adults. The results of these studies suggest that 1) the greatest quantity of CD34(+)CD38(-) and CD34(+)DR(-) cells was found in fetal and infant bone marrow, 2) the frequency of cycling CD34(+) cells declines with maturation and aging, and 3) an age-dependent difference in lineage commitment occurs.