The ultimate goal of this project is to cure Sickle Cell Disease (SCD) by autologous bone marrow transplantation after genetic alteration of hematopoietic stem cells with Adeno-Associated Virus (AAV) vectors. The AAV vectors will carry specially designed, anti-sickling beta-globin genes (BAS) which encode polypeptides that inhibit sickle hemoglobin (HbS) polymer formation and, therefore, inhibit erythrocyte sickling. The BAS-globin genes will be regulated by Locus Control Region (LCR) sequence that direct high level os human globin expression specifically in erythroid cells. The minimal LCR sequences required for high level expression will be determined by constructing various AAV HS 2 BAS-globin vectors and assaying these constructs in transgenic mice. Constructs that direct high levels of expression in this stringent assay will be utilized to produce virus. Initially, the AAV HS 2 BAS-globin viral stocks will be used to infect human marrow that will be maintained in the long term culture-initiating cell (LTC-IC) assay as described in Project 2. The efficiency of infection of hematopoietic stem cells will be assessed by determining the fraction of LTC-C that form BFU-E (burst Forming Units-Erythroid) containing BAS-globin sequence. BAS-globin polypeptides will also be quantitated in BFU-E derived from LTC-IC. When conditions required for efficient infection of stem cells and high level expression of transferred globin genes are defined, marrow from SCD patients will be infect. BFU-E derived from LTC-IC of SCD marrow will be assayed for BAS- globin DNA and polypeptides. BAS activity will be quantitated by expanding erythroid progenitors in Fibach's liquid culture system and measuring the inhibition of HbS polymer formation in hemolysates prepared from these cells. Also, erythroid cells from expanded liquid cultures will be deoxygenated in vitro to evaluate anti-sickling effects. When the anti-sickling properties of transferred globin genes are demonstrated in vitro, clinical trials will be initiated. Initially, only patient s with HLA matched allogeneic donor will be transplanted wit infected, autologous marrow so that a viable alternative therapy is available. The conditions for infection will be identical to those determined for efficient infection of the long term marrow cultures described above; however, the experiments will be scaled up for gene therapy. Blood samples will be obtained each week post-transplantation and the levels of BAS polypeptides will be quantitated. Anti-sickling activity will be assessed by measuring the inhibition of HbS polymer formation in hemolysates prepared from erythroid cells that are obtained from expanded cultures. Ultimately, the efficacy of the therapy will be determined by the degree of correction of the severe pathology of SCD.
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