The long-term goal of the project is to ameliorate or cure sickle cell disease and beta-thalassemia (thal) by retroviral gene transfer of normally functioning human beta or gamma globin genes into the hematopoietic progenitor cells (HPC) including stem cells of patients with these disorders. Retroviral vectors containing these globin genes and their control elements such as the locus control region (LCR) will be used to transduce human HPC from bone marrow or peripheral blood progenitor cell (PBPC) harvests. Ultimately, the goal is to cure the patients by autotransplantation by harvesting of HPC from patients with sickle cell disease and beta-thal, transducing these cells to restore high level gamma- or beta-globin expression, and then re-infusing the gene-corrected cells back into the patients. Progress has been made over the past five years in 1) the construction of beta and gamma globin gene containing retroviral vectors that are stably transmitted into target murine HPC; and 2) the conditions or transferring and expressing human genes such as the human multiple drug resistance (MDR) gene in murine and human HPC. In studies in this grant, improved human globin gene containing vectors will be constructed and tested in murine and human HPC, and more efficient methods of transferring and expressing these genes while maintaining their long-term repopulating ability will be explored. These methods will include the use of long-term marrow culture, and purified HPC with stroma and/or new growth factor combinations. In addition, the human MDR CDNA will be added to globin gene containing vectors to provide a selectable marker that can be used to enrich for globin gene transduced HPC in vitro and in vivo. Conditions which favor the engraftment and expression of globin gene transduced HPC without marrow ablation in murine models of sickle cell disease and beta-thal will be sought as well. Lastly, when the PI has obtained appropriate human globin gene containing vectors and culture conditions for their transduction and expression in murine and human HPC, we plan to design and initiate phase 1 clinical trials in sickle cell and beta-thal patients to test the safety and efficacy of retroviral globin gene transfer as an approach to the treatment of these disorders.
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