Study Of Gene Therapy For Hemophilia B

Hemophilia B is an inherited serious bleeding disorder due to a partial or complete deficiency of clotting factor IX activity in the plasma. Conventional therapy depends on infusion of human plasma or thrombinogen complex . The therapeutic effect is temporay and periodic infusions are required. In addition, such therapy is associated with significant risks of AIDS and hepatitis. Thus, it appears beneficial to develope an alternative therapy for hemophilia B. One model has been the use of recombinant retrovirus containing human factor IX cDNA to transduce fibroblasts as the target cells for gene transfer.In this study, we have constructed a series of recombinant retroviral vectors and plasmids in which human factor IX cDNA is driven by different promoters. These vectors includes: LIXSN vector (LN series, the FIX cDNA driven by LTR promoter), LNCIX vector (LN series, the FIX cDNA driven by CMV promoter), LCIXSN vectors (LN series, the FIY cDNA driven by LTR and CMV promoters), G1NaCIX vectors (G1Na series, the FIX cDNA driven by CMV promoter), G1NaC-I-IX vectors (G1Na series, the FIX cDNA containing IL-2gene intron and driven by CMV promoter), G1NaC-I'-IX vectors (G1Na series, the FIX cDNA containing internal intron and driven by CMV promoter), G1NaC-K-IX vectors (G1Na series,the FIX cDNA containing the Kozak consensus sequence and driven by CMV promoter). MFG-IX(MFG backbone without selecting gene,FIX cDNA driven by LTR promoter), MP6-IX(MP6 backbone, the FIX cDNA containing IL-2 gene intron and driven by CMV promoter). These retroviral vectors and recombinant plasmids were transfected into various target cells, such as the packaging cell line PA317, the human fibrosarcoma cell. HT1080, the skin fibroblasts of mouse, rabbit and hemophilia B patients, the myoblasts of mouse and human, the bone marrow cells of mouse and human and the bone marrow stroma cells of hemophilia B patient with different efficiency transfection and expression level. Many factors affecting the FIX cDNA expression in vitro such as double...