704. Ex vivo and in vivo expansion of HoxB4-Transduced CD34+ cells from dogs, baboons, macaques and humans

Successful ex vivo and in vivo expansion of transgene modified hematopoietic stem cells (HSC) have great application potential for clinical gene therapy. Overexpression of the human HoxB4 gene induces ex vivo expansion and self-renewal of murine HSC. Since mouse and human differ substantially, we have started to study the effect of HoxB4 on HSC in a large animal model. Here we investigated if human HoxB4 enhances ex vivo expansion and in vivo selection of CD34+ cells from mobilized dog peripheral blood (PB), cytokine primed baboon and macaque bone marrow (BM), human cord blood (CB) and mobilized human PB. CD34+ cells were transduced with Phoenix-GALV or RD114-pseudotype MSCV-HoxB4-ires-GFP or the control vector, MSCV-ires-YFP. After 4 weeks culture, the percentage of HoxB4-overexpressing cells significantly increased for dog, baboon and macaque cells. HoxB4 also imparts a noteworthy effect on expansion of progenitors. HoxB4 expression resulted in an up to 2-fold expansion for human PB, 4-fold expansion for human CB, 5 to 7-fold expansion for baboon and macaque, and 28-fold expansion for dog cells in CFUs when compared with YFP-transduced cells. Furthermore, in vivo competitive repopulation experiments in NOD/SCID mouse model were performed to test the effects of HoxB4 on HSC. The engraftment level for GFP+ cells was 6-fold higher for PB cells, and 10-fold higher for CB cells compared with YFP+ cells, indicating a significant growth advantage of HoxB4-transduced cells. To examine whether HoxB4 expression promotes expansion of HSC in large animal model, myeloblated dogs were transplanted with HoxB4- and YFP- transduced cells. Preliminary data show that the ratio of GFP+ cells to YFP+ cells increased from 0.9 at day 0 to ratio of 9 at day 20 post-transplant, demonstrating a remarkable in vivo selection of HoxB4-transduced cells in clinically relevant model. To explore the mechanisms that underlie the apparently more pronounced effects on cells from dog, macaque and baboon compared to human cells, we monitored the HoxB4 expression in these cells at the RNA level by real-time RT-PCR and at the protein level by western blot. Comparable level of HoxB4 and YFP transcripts was detected in dog and mouse transduced cells, while copies of HoxB4 transcripts were only 10–30% those of YFP transcripts for human and baboon transduced cells. Likewise, HoxB4 protein expression in human and baboon cells decreased continuously with time, in parallel with the differentiation of these cells. However, protein expression in transduced mouse and dog cells was stable, which was associated with inhibition of cell differentiation. We also found that HoxB4 was tyrosine phosphorylated in transduced mouse and dog cells. Our results suggest that the higher level of HoxB4 protein expressed inhibit the differentiation and promote self-renewal of HSC, while lower level confers a less pronounced effects on self-renewal. Further insights into the regulation of transduced HoxB4 in human cells might pave the road to its successful application in clinical gene therapy.