69. Selection of Hematopoietic Progenitor and Stem Cells in Transplanted Rhesus Macaques Using MGMT and HOXB4 Lentiviral Vectors
Authors: Yan Shou, John Gray, Brian Agricola, Zhijun Ma, Geeta K. Nair, Clinton F. Stewart, Derek A. Persons, Brian P. Sorrentino
Journal: Molecular Therapy (2006)

Gene transfer of O6-methylguanine-DNA-methyltransferase (MGMT) into HSCs results in efficient in vivo selection of murine HSCs after treatment with temozolomide (TMZ) and O6- benzylguanine (BG). In addition, HOXB4 coexpression is associated with an increase in HSC selection efficiency using a DHFR drug selection system in mice. Based on these results, we have studied whether the MGMT and HOXB4 selection systems are efficacious in a rhesus macaque autologous transplantation model. SIV vectors were constructed incorporating either MGMT alone or MGMT together with HOXB4 in a GFP or YFP backbone. In our first transplant case, we purified CD34+ cells from mobilized bone marrow cells and split the graft into two parts: half of the cells were transduced with a SIV MGMT-GFP vector and half were transduced with a SIV MGMT-HOXB4-GFP vector. In our second and third transplant cases, half of the CD34+ cells were transduced with a SIV MGMT- YFP vector and the other half with a SIV MGMT-HOXB4-GFP vector. During the early engraftment period, there was a slight advantage (2-fold maximal) for HOXB4 transduced cells relative to the "MGMT-only" vector. However, by 1 month post- transplantation, there was no difference in marking between the two vectors, suggesting HOXB4 had little effect on long-term repopulating cells. The first animal has been treated with 7 courses of TMZ/BG, starting with a fixed dose of 120 mg/m2 for BG and 170 mg/m2/day for 5 days for TMZ. The dose of TMZ was escalated on each course, and well tolerated by the animal. For the 7th course, the animal received 450 mg/m2 of TMZ with BG, a significant dose increase relative to the pediatric maximum tolerated dose (185 mg/ m2 without BG in irradiated patients). This increase in dose intensity was verified by formal pharmacokinetic measurements. After each course of drug treatment, there was a rapid increase in GFP marking in all lineages. The highest marking achieved after last course of TMZ/BG treatment was 91% for myeloid cells and 50% for lymphoid lineages. However, the proportion of GFP+ cells subsequently declined and leveled off at 13% for granulocytes, 17% for B cells and 36% for T cells. The second animal had been treated with two courses of TMZ/BG and there was about 7-fold increase in GFP marking in granulocytes and a 3 and 8-fold increase in B, T cells respectively. Altogether, we have shown that TMZ selection was occurring mainly in progenitor cells with limited self-renewal capacity and to a lesser degree at the HSC level as evidence by increased baseline GFP marking. We were also able to achieve a significant increase in dose intensity in the first animal, and to successfully administer multiple courses of drug treatment. These results suggest a clinical strategy for hematopoietic protection in cancer treatment. We are now treating these animals with BCNU plus BG to determine if HSC selection efficiency is increased relative to TMZ. Lastly, we did not see evidence of HSC amplification with HOXB4 expression, suggesting that the robust selection seen in mice is relatively species specific.