Reduced Intensity BCNU/O6-BG Selection of MGMT-P140K Transduced Hematopoietic Stem Cells Is Efficient at Reduced Toxicity and Leads to an Unrestricted Oligoclonal Differentiation of Modified Murine Repopulating Stem Cells

• Published in: Blood 2004-11, Vol.104 (11), p.2109-2109
• Main Authors: Ball, C., Schmidt, M., Pilz, I., Fessler, S., von Kalle, C., Glimm, H.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V104.11.2109.2109

Successful selection of gene modified hematopoietic stem cells against the majority of non-modified cells may increase the efficiency and safety of clinical gene therapy. Especially if a reduction of the intensity of the myelotoxic pre-transplant conditioning treatment is sought, selection of the initially low percentage of retrovirally gene modified stem cells is required. In addition to the decreased drug related toxicity, such a procedure reduces the risk of genotoxicity caused by insertional mutagenesis simply by diminishing the likelihood of transplanting stem cells that carry unwanted insertion sides compared to strategies that attempt to increase efficiency by increasing vector dose and/or the number of engrafted gene-modified cells. To date, the mutant O6-methylguanine-DNA methyltransferase (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, that is associated with very considerable toxicity. 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 (1x105 and 4x105) 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 (either 10 mg/kg O6-BG and 2,5 mg/kg BCNU or 20 mg/kg O6-BG and 5 mg/kg BCNU) as compared to the commonly used regimens. 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 5 rounds of selection. 3 of 48 mice died during the selection procedure. Although in 97% of all mice GFP+ blood cells were detectable 4 weeks after transplantation, an efficient selection (>twofold increase in transduced cells) was only seen in mice with >3% GFP+ initial blood cells. 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 hematopoiet