Pre-clinical and clinical studies on gene therapy of sickle cell disease and beta thalassemia. The goal of this project is to perform the pre-clinical and clinical studies that are required for the eventual application of the therapeutic gene transfer in sickle cell disease and beta thalassemia. Specifically, 1) stem cell mobilization is required for the application of ex vivo stem cell gene therapy but the current approach based on administration of G-CF cannot be used in sickle cell disease because the resultant leukocytosis precipitates a sickle cell crisis with severe (or lethal) consequences. We will develop new approaches of mobilization that can be used safely in sickle cell disease. Two clinical studies will be done. The first is based on the assumption that recovery from the cytotoxic effects of hydroxyurea results in hematopoietic cell regeneration that is accompanied by increased rate of mobilization of stem/progenitor cells. The second approach is based on the evidence that mobilization using anti-VLA-4 antibody or Flt-3 ligand is characterized primarily by release of progenitor cells and small degree of leukocytosis. Preclinical studies using a combination of anti-VLA-4 with hydroxyurea, or anti-VLA-4 with Flt-3 ligand will be done in baboons to identify a treatment scheme that will result in optimal mobilization with minimal change in white blood cell numbers. The optimal treatment scheme will subsequently be applied to patients with sickle cell disease. 2) We will perform pre-clinical studies aimed to determine whether MLV-based """"""""insulated"""""""" betagamma globin gene vectors can be used for clinical studies in sickle cell disease and beta thalassemia. Specifically, we will a) test whether such vectors can correct the defects in sickle cell and beta thalassemic mice; b) assess transduction rates, levels of expression of the transferred betagamma globin gene and vector toxicity in the baboon transplantation model; c) determine betagamma gene expression and transduction rates in primary human erythroid cells following transduction of BFU-E of patients with sickle cell disease or beta thalassemia; d) measure transduction rates of human stem cells in the SCID/NOD murine model. 3) Successful completion of these pre-clinical studies will allow us to proceed with a clinical study in patients with Hb S disease or beta thalassemia. a) Clinical grade vectors will be produce, b) the necessary regulatory requirements will be fulfilled, c) a clinical study of three patients with sickle cell disease and six with beta thalassemia will be initiated to test safety and function of globin gene vectors.
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