913. MGMT (P140K) Allows for Safe, Efficient, and Sustained In Vivo Selection and Chemoprotection in a Large Animal Model
Authors: Brian C. Beard, Tobias Neff, Laura J. Peterson, Bobbie Thomasson, Jesse Thompson, Hans-Peter Kiem
Journal: Molecular Therapy (2004)

In vivo selection of genetically modified cells has the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. We have previously reported successful in vivo selection of MGMT overexpressing cells in a clinically relevant canine model of allogeneic and autologous stem cell transplantation. Here we describe long-term stable in vivo selection and chemoprotection using the MGMT system. CD34-enriched donor marrow cells were transduced with an RD114-pseudotype retroviral vector encoding P140K and eGFP. Transduced cells were infused into two myeloablated DLA-identical allogeneic recipients or two autologous recipients. After stable engraftment four dogs were treated with various regimens of O6BG and BCNU or O6BG and temozolomide. Initially, the dose of 5 mg/kg O6BG and 0.4 mg/kg BCNU increased marking, but also caused dramatic myelosuppression. After blood cell counts recovered an identical dose of O6BG and BCNU achieved additional selection with no myelosuppression demonstrating marrow protection. The pronounced myelosuppression initially observed in the first dog was alleviated in the second dog by using slowly escalating doses of BCNU with more frequent treatments before further dose escalation. As an alternate selection strategy temozolomide was used in place of BCNU in the two autologous dogs. Initially, low doses of temozolomide followed by dose escalation of up to 600 mg/m2 and 700 mg/m2 has shown efficient in vivo selection without myelosuppression. The chemoprotection of bone marrow in all dogs appears to be very durable and protects against myelosuppression at chemotherapy doses that lead to pronounced cytopenia in control animals. Gene marking in hematopoietic repopulating cells was assessed by flow cytometry and real-time PCR. In vivo selection was observed in all lineages with granulocyte marking of up to 89% with O6BG and BCNU and 98% with O6BG and temozolomide. The marking is very durable with follow up after selection to 20 months. Morphological analysis of marrow aspirates 12 months and 7 months after selection showed no evidence of myelodysplasia. In an attempt to characterize the clones contributing to long-term hematopoiesis linear-amplification mediated PCR was carried out on peripheral blood DNA. Integration analysis has shown dogs are polyclonal before and after selection with no trend towards oligoclonal or monoclonal states. To probe the absolute number of provirus in clones contributing to long-term hematopoiesis real-time PCR analysis was carried out on peripheral blood DNA. The provirus copy number was less than two copies per cell. Our data demonstrate that less than two provirus copies are sufficient to protect clones from chemotherapy. In summary our data from a clinically relevant canine model demonstrate that MGMT(P140K)-transduced cells can be safely, efficiently and durably selected with a follow up of up to 20 months, and that the high marking levels attained by in vivo selection provide effective marrow protection. Finally, using P140K gene-marked stem cell transplants in conjunction with either BCNU or temozolomide for treatment of solid tumors would alleviate the dose limiting myelosuppression often seen with such treatments.