Application Of MGMT Gene In Platelet-specific Gene Therapy For Hemophilia B

Objective:In the process of platelet targeted gene therapy of hemophilia B, hematopoietic stem cells are modified by F9 gene in vitro and subsequenctly transplanted into recipients. It is difficult to achieve 100% of the hematopoietic stem cells are effectively modified by the target gene. During bone marrow transplantation, although recipient mice are lethally irradiated, the native hematopoietic stem cells still can not be thoroughly cleared. The transplanted allogeneic cells would face the competition of the native cells. Therefore, there are only part of hematopoietic stem cells successfully expressed the target gene after transplantation. This will affect the expression level and the long-term stability of the target gene. In order to improve the expression level of the target gene and the reconstitution of the transplanted cells, we explored to use MGMT (P140K), a drug resistent gene, to increase the proportion of gene-corrected hematopoitic stem cells in vivo.Methods:We cloned MGMT gene from a human hepatocyte cell line, and changed proline (CCC) to lysine (AAG) at site 140 by site-directed mutagenesis method. The acquired MGMT (P140K) gene were then subcloned into vector pWPT2bF9, which was used to produce the lentivirus 2bF9-LV (P140K). Cells were transduced with 2bF9-LV (P140K), and a combination of carmustine (BCNU) and 06-BG was used fro cells selection. With the treatment of the two drugs, only the cells received and express MGMT (P140K) would survive the treatment.Results:We successfully constructed pWPT2bF9-MGMT (P140K) expression vector. With this vector we produced lentivirus 2bF9-MGMT (P140K)-LV. We also made a GFP expressing lentivirus GFP-LV. The titer of lentivirus can reach 109 copy number/ml. GFP-LV was used to transduce 293T cells and Dami cells, the expression of GFP was detect by FACS.2bF9-MGMT (P140K)-LV was transduced Dami cells, and the expression of FIX in cells was demonstrated by FIX ELISA, We also tested the function of MGMT (P140K) in 293T cells, and the cells expressed MGMT (P140K) survived the drug challenge.Conclusion:We used the lentiviral packaging system produced two lentivirus 2bF9-MGMT (P140K)-LV and GFP-LV. The expression of GFP and MGMT were demonstrated in the transduced 293T cells and Dami cells. We also established drug screening system using BCNU and O6BG, MGMT (P140K) expressed cells successrully survived the drug selection. This provides a reliable system for future in vivo hematopoitic stem cell selection and improve the efficiency of gene transfer of bone marrow transplantation.