1055. Reduced Toxicity Selection of Modified Murine Repopulating Stem Cells Results in an Unchanged Differentiation Potential of Oligoclonal MGMT-P140K Overexpressing Murine Hematopoiesis
Authors: C. Ball, M. Schmidt, C. von Kalle, H. Glimm
Journal: Molecular Therapy (2005)

Efficiency and safety of clinical gene therapy may be increased by a successful selection of gene modified hematopoietic stem cells against the majority of non-modified cells. A reduction of the intensity of the myelotoxic pre-transplant conditioning treatment requires an efficient selection of the initially low percentage of retrovirally gene modified stem cells. In addition to the decreased drug related toxicity, such a procedure reduces the risk of genotoxicity caused by insertional mutagenesis by diminishing the likelihood of transplanting stem cells that carry unwanted insertion sides. To date, the mutant MGMT enzyme that confers resistance to nitrosoureas such as BCNU is the drug-resistance gene that allows most efficient selection at the stem cell level. In the murine model, MGMT selection by BCNU and O6-BG has mostly been performed at LD 50 dose levels of the selecting agents. We now established minimal dosage requirements of transplanted cells as well as of BCNU and O6-BG that allow an efficient selection of murine long-term hematopoiesis. Bone marrow cells from 5-FU treated C57 BL/6J mice were transduced with an MGMT/IRES/eGFP encoding retroviral vector and transplanted at two dilutions (1105 and 4105) into 84 lethally irradiated syngeneic recipient mice. Starting 4 weeks post-transplant, the mice were treated monthly with two reduced dosages of O6-BG and BCNU (10 mg/kg O6-BG and 2,5 mg/kg BCNU) as compared to the commonly used application rates (20 mg/kg O6-BG and 5 mg/kg BCNU). Kinetics, differentiation and clonality of the transduced hematopoiesis were monitored by FACS and LAM-PCR analysis of serial peripheral blood samples over a total of 7 rounds of selection. 3 of 48 mice died during the selection procedure. The percentage of transduced hematopoiesis in 9 of 24 mice treated with the higher BCNU and O6-BG dosage increased at least twofold as compared to 5 of 24 treated with the lower dosage and 1 of 24 mice without selection. Using Poisson statistics, the frequency of selectable hematopoietic units was calculated to be as low as 1 per 5105 cells transplanted. Using LAM-PCR, we were able to detect stable oligoclonal hematopoiesis after transplantation of 4105 cells for more than 6 months, suggesting that a subfraction of repopulating cell clones present were not selectable, or that the contribution of individual clones was small. No differences in lineage differentiation of the GFP+ hematopoiesis as determined in lineage marker expression by FACS could be observed after selection as compared to unselected control mice. Investigation of recently performed secondary transplantations indicates that the transduced hematopoietic cells actually maintain stem/progenitor cell characteristics. In summary, our results demonstrate that a reduced BCNU and O6-BG with lower toxicity allows selection of MGMT expressing murine hematopoietic stem cells with unperturbed multilineage differentiation potential. Moreover, under these conditions, a subfraction of transduced repopulating cell clones was selectable.