Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

In an attempt to develop efficient procedures of human hematopoietic gene t herapy, retrovirally transduced CD34(+) cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced gra fts. Samples were prestimulated on Retronectin-coated dishes and infected w ith gibbon ape leukemia virus (GALV)-pseudo-typed FMEV vectors encoding the enhanced green fluorescent protein (EGFP), Periodic analyses of bone marro w (BM) from transplanted recipients revealed a sustained engraftment of hum an hematopoietic cells expressing the EGFP transgene, On average, 33.6% of human CD45(+) cells expressed the transgene 90 to 120 days after transplant ation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45(+) cell s expressing the EGFP transgene at this time. The transplantation of purifi ed EGFP(+) cells increased the proportion of CD45(+) cells positive for EGF P expression to 57.7% at 90 to 120 days after transplantation. At this time , 18.9% and 4.3% of NOD/SCID BM consisted of CD45(+)/EGFP(+) and CD34(+)/EG FP(+) cells, respectively. Interestingly, the transplantation of EGFP(-) ce lls purified at 24 hours after infection also generated a significant engra ftment of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, suggesting that a numb er of transduced repopulating cells did not express the transgene at that t ime. Molecular analysis of NOD/SCID BM confirmed the high levels of engraft ment of human transduced cells deduced from FAGS analysis. Finally, the ana lysis of the provirus insertion sites by conventional Southern blotting ind icated that the human hematopoiesis in the NOD/SCID BM was predominantly ol igoclonal. (C) 2000 by The American Society of Hematology.