The ultimate goal of our proposal to develop high efficiency retroviral vectors capable of delivering the human beta-globin gene into whole animals is to demonstrate the feasibility of somatic cell gene therapy for human hemoglobinopathies. Various parameters required for efficient introduction, regulated expression and potential for gene therapy will be considered. 1) High Efficiency Vectors Carrying the Human Beta-Globin Gene: High titre recombinant retroviruses will be generated capable of stable, accurate expression of the human betaglobin gene as well as vectors which lack enhancer/promoter elements and splice junctions as these may interfere with appropriate expression. Expression from the natural promoter in an inverted orientation relative to the transcription of the retrovirus genome and the expression of the cDNA from the retroviral promoter will be pursued. Helper-free packaging cell lines will be used to generate high titre recombinant virus. 2) Cell culture model for erythroid differentiation: The virus constructs will be tested for efficiency and regulation of beta-globin gene expression in erythroleukemia cells to insure that subsequent experiments are carried out using recombinant viruses capable of expressing intact beta-globin RNA in a regulated manner. 3) Bone marrow infection: A major goal is to introduce the human beta-globin gene into pluripotent stem cells via retroviral vectors. Both in vitro long-term bone marrow culture and in vivo bone marrow transplantations will be carried out. We also plan to infect human bone marrow cells for long-term bone marrow cultures in vitro. 4) The mouse beta thalassemia model: After successful establishment of human betaglobin gene expression in normal mice, homozygous beta thalassemic mice will be tested. The effects of the expression will be monitored and the prospects for long-term maintenance of the retrovirus-containing stem cells will be assessed. 5) The human alpha-globin gene: Retrovirus vectors carrying the human alpha-globin gene will be constructed, tested for expression and introduced into the bone marrow of normal mice and, ultimately, heterozygous alpha-thalassemic mice. 6) Germ line transfer: We plan to infect preimplantation embryos with retroviral vectors in order to study the expression of globin genes carried in the germ line of normal and thalassemic mice.
| Li, C L; Dwarki, V J; Verma, I M (1990) Expression of human alpha-globin and mouse/human hybrid beta-globin genes in murine hemopoietic stem cells transduced by recombinant retroviruses. Proc Natl Acad Sci U S A 87:4349-53 |
| Axelrod, J H; Read, M S; Brinkhous, K M et al. (1990) Phenotypic correction of factor IX deficiency in skin fibroblasts of hemophilic dogs. Proc Natl Acad Sci U S A 87:5173-7 |
