Differential Effects of HOXB4 Overexpression on Short and Long-Term Repopulating Cells in Nonhuman Primates.

The inability to expand hematopoietic stem cells (HSCs) has been a significant limitation for clinical transplantation and gene therapy applications. Here we examined in a clinically relevant nonhuman primate model the ability of HOXB4 to expand HSCs and thus potentially overcome this limitation. Using a competitive repopulation assay we directly compared engraftment of HOXB4-transduced and control-transduced CD34⁺ cells. In 3 animals, cells were infused after a 3-day transduction and in 2 animals after an additional 6 to 9 days ex vivo expansion. Follow-up for these animals is up to 15-months. In the 3 animals that received HOXB4GFP-transduced cells without additional ex vivo culture, gene transfer efficiencies in CD34⁺ cells were similar between HOXB4GFP and YFP transduced cells: 45% (range 36–55%) vs. 38% (range 36– 40%). We observed a dramatic increase in HOXB4GFP marked cells from 20–30% to 52–62% during the early engraftment period, resulting in an up to 10-fold difference in granulocyte marking between HOXB4GFP and YFP marked cells at 5 weeks post-transplantation. Although gene-marking levels declined over time, HOXB4 marking was still about 2 to 3-fold higher than marking in control cells even at 15 months post-transplantation. A more pronounced effect was observed in the 2 animals that received HOXB4-overexpressing cells after an additional 6 to 9 days of ex vivo culture. Again, no difference in transduction efficiency was observed between YFP (range 34–49%) and HOXB4GFP (range 39–43%) marked cells before transplantation. However, HOXB4 marking was higher than YFP marking 1 week after transplantation, with up to a 34-fold difference in granulocyte marking at 2 weeks post-transplantation. Although the difference was decreased thereafter, a 4 to 10-fold difference was maintained in granulocyte marking after 3 months post-transplantation, suggesting a potential effect on the expansion of long-term repopulating cells. Subset analysis by flow cytometry and Taqman PCR showed HOXB4GFP and YFP marking in all subsets. Marking in CD13⁺ granulocytes and CD14⁺ monocytes was higher with HOXB4GFP-transduced cells and marking in CD3⁺ T cells was higher with YFP-transduced cells, suggesting that HOXB4 overexpression may have a more pronounced effect on engraftment and differentiation of myeloid than T-lymphoid precursors. LAM-PCR analysis demonstrated multiple clones of HOXB4GFP⁺ cells and control YFP⁺ cells. Our results demonstrate that HOXB4 overexpression in CD34⁺ cells has a very dramatic effect on expansion and engraftment of short-term repopulating cells with a less pronounced effect on long-term repopulating cells. These data should have important implications for the expansion and transplantation of HSCs, in particular for cord blood transplantations.