Analysis of human CD45⁺engraftment and EGFP expression in human hematopoietic cells in NOD/SCID mice.

Transduction efficiency of SCID repopulating cells (SRCs) by a single exposure of cord blood CD34⁺ cells to RD114-pseudotyped vector particles preloaded on retronectin after only 24 hours or 48 hours of preactivation was assessed over a series of experiments (Experiment 1, open triangles; Experiment 2, closed triangles; Experiment 3, open circles). Cells were cultured for a total of 96 hours before injection into recipient animals. After 8 weeks in vivo, the animals were killed and analyzed for human engraftment (greater than 0.5%) based on CD45⁺ expression (A) and functional marking based on EGFP expression (B). The number (N) of animals in each group is indicated. Engraftment was notably decreased in animals that received CD34⁺ cells transduced at 24 hours in vitro with RD114/MGirL22Y vectors, but this effect was not as significant in animals that received cells transduced at 48 hours. Remarkably, all animals that received RD114/MGirL22Y-transduced CD34⁺ cells engrafted with EGFP⁺ cells. Indeed, the best engrafted animals also had the highest level of EGFP⁺ cells. Control animals that received untransduced cells or cells either transduced by preloaded am/MGirL22Y vectors alone (*) or transduced by preloaded am/MGirL22Y particles at 24 hours, followed by exposure to viral supernatant at 48 hours and 72 hours, engrafted only with unmarked human cells (despite the presence of 10% serum in the conditioned medium).