Pharmacologic Control of HOXB4-Mediated Expansion of Murine Hematopoietic Stem Cells; Evidence for a Restricted Time Interval for Expansion.

Overexpression of the homebox transcription factor HOXB4 can enhance self-renewal of murine hematopoietic stem cells (HSCs) and thereby result in an increased number of HSCs in vivo. In mice transplanted with bone marrow cells transduced with a retroviral vector expressing HOXB4, HSC expansion stopped after HSC numbers regenerated to a normal level. Furthermore, when transduced bone marrow cells from primary transplant recipients were transplanted into secondary recipients, HSCs failed to recover to normal numbers (G. Sauvageau et al, Genes and Dev, 9:1753, 1995). One possible explanation for these results is that HSC expansion could be limited to an early time interval in the primary transplant recipient. In order to determine if a time-window exists for HOXB4-mediated HSC expansion, and to develop a method to control HSC expansion for gene therapy applications, we generated a retroviral vector expressing a HOXB4 protein that was fused to a variant estrogen responsive binding element (ERT 2 ). This HOXB4-ER T2 protein allowed HOXB4 function to be regulated with 4-hydroxytamoxifen (TAM). Murine bone marrow cells were transduced with the MSCV- HOXB4-ER T2 -GFP vector and transplanted into lethally irradiated recipients. A 3 week course of daily TAM treatment was started either immediately after transplant, or in a second cohort, 12 weeks after transplant. When TAM treatment was administered for the first 3 weeks after transplant, there was a 7-fold increase in the percentage of GFP positive peripheral blood leukocytes compared to the cohort transplanted with the same cells but not receiving TAM treatment (15% +/−8, n=7, VERSUS 2 % +/− 2, n=9). In contrast, an identical 3-week course of TAM treatment beginning at 12 weeks post-transplant had no effect on the proportion of GFP+ cells in the peripheral blood (3% +/−2, n=5 VERSUS 2% +/−2, n=4). Bone marrow cells from mice in each of these cohorts were harvested at 21 weeks after transplant, and infused into secondary recipients. The proportion of GFP+ blood cells noted in the primary recipients that were treated with TAM for weeks 1 through 3 was maintained in untreated secondary recipients, confirming that early TAM treatment had resulted in amplification at the level of HSCs. The other half of these secondary recipients was treated immediately after transplant with the same 3 week course of daily TAM treatment. TAM treatment in secondary recipients did not lead to a further increase in the proportion of GFP + blood cells compared to values in the untreated secondary recipients (9% +/−7, n=9 VERSUS 10% +/−3, n=6). These results show that the early 3 week time interval for HSC expansion was not reset with secondary transplantation and suggest that there is a HSC intrinsic mechanism that limits HOXB4-mediated expansion based on past replication history. This model would explain the physiologic limitation on HSC expansion that has been noted with wildtype HOXB4 vectors. Experiments are now in progress to further elucidate this putative mechanism, including further refinement of the time limits for expansion and microarray analysis of downstream target genes at different time points relative to transplantation.