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Modeling gene therapy of beta-Thalassemia : an evaluation of sub-lethal myeloablation and bone marrow transplantation as a curative approach Cavilla, Benjamin

Abstract

Beta-Thalassemia (β-Thalassemia) is a serious disease of the blood that leads to significant morbidity and mortality world-wide. It is caused by a heritable defect in hemoglobin synthesis that leads to accelerated red blood cell destruction, anemia and a plethora of secondary sequelae. While β-Thalassemia can be treated on an out-patient basis, effective control of disease symptoms via blood transfusion is burdensome, both emotionally and financially. Traditionally, the only curative measure has been bone marrow transplantation from a compatible donor. However, recent advances in the field of retroviral gene therapy have made the long-term correction of β-Thalassemia by autologous hematopoietic stem cell (HSC) transplant, a very real possibility. Even so, the risk of death associated with bone marrow transplantation procedures is still unacceptably high in many cases. The ultimate goal of the work described in this thesis was to explore the use of sublethal myeloablation in combination with bone marrow translantation as a curative approach to β-Thalassemia in the Hbb[sup th(d3)]/Hbb[sup th(d3)] mouse model. The results described in this thesis confirm the potential of hematopoietic stem cells to engraft in normal mice (B6C3 and C57BL/6J) in both the completely unirradiated and sublethally irradiated setting. Engraftment levels in the sub-lethally irradiated setting were linearly related to preparative radiation doses over the range 200 cGy (Rads per minute) to 500 cGy. In addition, engraftment levels were found to demonstrate a curvilinear relationship to the number of cells transplanted over the range 1 x 10⁴ to 1 x 10⁸ cells. Increasing radiation doses from 200 cGy to 300 cGy had the effect of increasing the maximal level at which engraftment levels plateau, decreasing the cell dose at which engraftment efficiencies begin to taper-off and decreasing the threshold after which engraftment levels increase linearly with cell doses. A mathematically-based model has been proposed to explain this relationship. It has been postulated that observed levels of engraftment are dependent upon the ratio of donor to recipient HSCs within the animal. As an alternative to decreasing the number of recipient HSCs by increasing preparative radiation doses, an attempt was made to increase the number of HSCs in the transplant inocula, by treating donors with 5-Fluorouracil prior to bone marrow harvest. However, this approach provided no additional engraftment advantage over bone marrow from untreated controls. Finally, mouse models of β-Thalassemia were transplanted with 3 x 10⁶ normal cells following sub-lethal radiation doses over the range 100 cGy to 500 cGy. In an attempt to model the conditions of gene therapy where genetic correction of HSCs can be as low as 60%, these cells were competed with 2 x 10⁶ syngeneic thalassemic cells. Mice treated with as little as 300 cGy showed signs of a therapeutic effect with marked improvements in all blood indices assessed. In addition, there was evidence of erythroid-specific amplification of normal red blood cells within thalassemic mice treated with preparative radiation doses of 300 cGy or higher. These results demonstrate that autologous bone marrow transplantation in combination with retroviral gene therapy techniques may be curative for β -Thalassemia in the sub-lethally irradiated setting with as few as 3 x 10⁶ corrected cells and 300 cGy of preparative radiation.

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