1039. Ex Vivo Pre-Selection Increases Marking Following Transplant of Mixtures of Bone Marrow from Wild Type and Green Fluorescent Protein-Methylguanine Methyltransferase P144K Fusion Protein Transgenic Mice

Abstract

Top of pageAbstract Methylguanine methyltransferase (MGMT) repairs alkylation of DNA resulting from 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) treatment. Mutant MGMT (MGMT|[ast]|) is resistant to inactivation by the irreversible inhibitor, O6-benzylguanine (BG), while retaining DNA repair activity. MGMT|[ast]| have been incorporated into retrovirus vectors for marking of hematopoietic stem cells, facilitating use of in vivo selection regimens that combine BG with BCNU or Temozolomide to enhance gene marking. Green fluorescent protein (GFP) is a convenient marker for monitoring transgene expression. We showed that GFP-MGMT|[ast]| fusion protein retains high fluorescence and MGMT|[ast]| activity when expressed by retrovirus vector transduced cells, and is a convenient model system to explore optimization of in vivo selection (Exp. Hematol. 32 (2004) 709). We engineered a GFP-MGMT|[ast]| fusion protein transgenic mouse (C57BL/6 oocyte injection; CMV enhancer/chicken ?-actin-promoter driven expression). All organs demonstrate strong GFP fluorescence with heterogeneity. With respect to blood cells, T-cells, B-cells, granulocytes, and Sca-1+ cells show 40%, 95%, 90%, and 70% green fluorescence, respectively. Transgenic mouse, but not wild-type mouse tissues retained high DNA repair enzymatic activity following 200?M BG treatment. Transgenic mouse bone marrow (BM) cells mixed with different ratios of wild-type BM cells were transplanted into myeloablated C57BL/6 mice. GFP fluorescent blood cells in transplanted mice were proportional to the ratio of transgenic to wild-type BM in the graft, where mice transplanted with 100% transgenic marrow demonstrated marking similar to that seen in transgenic mice. Transplanted mice were treated with BG/BCNU (20/6 mg/kg) two times 7 days apart. 3 weeks after selection, mice with baseline marking between 0.3% and 10% demonstrated 10-fold to 4-fold increase in marking, respectively. Because of potential in vivo toxicities, we also explored use of a single short time ex vivo selection (1 hour BG 40?M followed by addition of BCNU 80uM for 2 additional hours; cells washed before transplant) using 1:4, transgenic:wild-type mixture before transplantation (1|[times]|10exp6 BM cells). Animals transplanted with untreated mixtures showed 9|[plusmn]|2.2 % of GFP+ cells in blood 4 weeks after transplantation whereas ex vivo selected cells transplanted mice showed 68.8|[plusmn]|7.1 % (P< 0.0001). There was no significant difference in absolute white blood cell numbers. This demonstrates the feasibility of using a single short time ex vivo selective treatment with BG/BCNU to select for cells that express GFP-MGMT|[ast]|. There does not appear to be any requirement for either continued presence of the BG/BCNU or prolonged ex vivo growth of the cells to achieve selective enhancement of in vivo marking. Studies are in progress to determine whether this approach can also be used with retrovirus GFP-MGMT|[ast]| transduced BM cells. This also demonstrates the utility of GFP-MGMT|[ast]| transgenic mice to explore selection regimens.