The long-term goal of this project is to ameliorate or cure sickle cell disease by retroviral gene transfer of normal 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, 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 for transferring and expressing human genes such as the human murine HPC; and (2) the conditions for transferring and expressing human genes such as the human murine HPC; and (2) the conditions for 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 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 isolated sub-populations of HPC, long-term marrow culture and repeated transduction of HPC. 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 and expression of globin gene-transduced HPC without marrow ablation will be sought as well Lastly, when we have 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 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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