CB CD34⁺ cells at the indicated doses were preincubated for 96 hours in the presence of FL, TPO, SCF, and IL6; incubated overnight with vector particles (TU 50 × 10⁶/mL, or MOI 50): and further cultured ex vivo for a total of 4 weeks. Progeny cells were harvested and inoculated into sublethally irradiated NOD/SCID mice. Each mouse was killed 6 to 8 weeks later and the BM was harvested and assessed for levels of human engraftment, percentage of GFP-expressing human cells, and evidence of multilineage engraftment. Two to 10 million unfractionated BM cells from each primary mouse were inoculated into 3 secondary mice, which were killed 6 to 8 weeks later. The BM of secondary recipients was collected and analyzed as described for primary mice. Twenty million unfractionated BM cells from secondary recipients were inoculated into tertiary mice, which were killed 6 to 8 weeks later. The BM of tertiary recipients was collected and analyzed as described for primary mice.

ND indicates not done; —, not evaluable; NA, not applicable; and D, dead.

Number of copies of the vector per genome (mouse + human) obtained by RTQ-PCR as described in “Materials and methods”; in parentheses, the corresponding GFP⁺ population in the total (mouse + human) BM (eg, 0.02 means that a vector copy was likely expressed in 2% of human cells in secondary mouse A2.1, whereas by FACS, the percentage of GFP⁺ cells in the total BM was 1.7%).

Multilineage engraftment (ME) was defined by the presence of CD19⁺, CD34⁺, CD14⁺, CD33⁺, and CD41⁺ cells within the CD45 gate and by anti–glycophorin-A⁺ cells in total BM cells.