Developing a protocol in vivo for recombinant adeno-associated virus-mediated gene therapy of hemoglobinopathies

Document Type


Publication Date



Towards gene therapy for thalassemias and sickle cell disease this laboratory has constructed recombinant adeno-associated viruses (rAAVs) that harbored human α- and β-globin cDNAs/ genes, which efficiently expressed globin mRNAs and polypeptides in erythroid cells (Ohi, S. et al., Gene 89: 279, 1990; Dixit, M. et al., Gene 104: 253, 1991; Ohi, S. and Kim, B.C., Blood 84: 263a, 1994, J. Pharm. Sei., in press, 1995). As the test system, we are now developing a protocol(s) for gene therapy of a mouse model of β-thalassemia (C57BL/ using the rAAVs with a possible application to humans in the future. Mouse bone marrow hematopoietic stem/progenitor cells (BMHSCs) were purified/ enriched by Histopaque 1077 (Sigma, density 1.077 g/c.c.) density gradient centrifugation and immunomagnetic purging (Ohi, S. et al. FASEB J. 7: A1304, 1993). The L3T4-, Lyt2-, Thy 1.2+ cells had a capability to differentiate into BFU-E, CFU-GM, and CFU-GEMM in the methyl-cellulose clonogenic assay. In addition, when the host marrow cells were depleted by 70% by cyclophosphamidetreatment, about 10,000 purified BMHSCs restored the cell number of the host marrow in about a week. The rAAV vectors effected synthesis of human hemoglobin in the BMHSCs in culture. (Supported by grants from The American Heart Association-NCA and The Armstead-Barnhill Foundation for Sickle Cell Anemia).

This document is currently not available here.