Gene Therapy for BETA ZERO Thalassemia

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The goal of this project is to perform pre-clinical and clinical studies aimed to test the safety and efficacy of hemopoietic stem cell mobilization and of gene therapy in beta zero thalassemia major. Beta zero thalassemia, i.e. thalassemia with complete absence of beta chain synthesis, has been selected as the target disease because of the possibility to use efficient quantitative cytochemical and biochemical techniques to identify the newly produced beta globin chains and RNA in the patients' blood in spite of the presence of transfused red cells. The clinical studies will be done in Greece, because of the number of beta zero thalassemia major patients available for enrollment. Our Specific Aims are 1) To improve the beta globin gene lentiviral vector developed during the current funding cycle. We will complete already initiated studies aimed to produce a beta lentiviral vector also carrying an insulator element expected to decrease position effects and increase vector safety. 2) To determine the safety and efficacy of stem cell mobilization in patients with beta zero thalassemia major. Three protocols will be used. Non-splenectomized (6) patients pretreated with hydroxyurea and subsequently mobilized with G-CSF; non-splenectomized (6) or splenectomized (6) patients treated only with G-CSF. 3) To determine the safety and efficacy of a gene transfer protocol consisting of HSC mobilization, patient conditioning using melphalan and autotransplantation with lentiviral vector transduced CD34+ cells. Twelve patients will be treated with Melphalan in a dose escalation scheme (70, 100, 120 mu g/m2) . Four patients will not receive conditioning and serve as controls. 4) To determine beta globin gene transfer rates and persistence of beta globin gene expression in patients transfused with the transduced CD34+ cells. Posttransplant analysis of vector expression will be based on quantitation of A-reticulocytes, measurements of beta/alpha globin mRNA ratios in the blood by quantitative PCR and analysis of globin gene expression in erythroid progenitor cell colonies. 5) To investigate the integration biology of the transferred lentiviral vectors. Using genomic techniques we will 1) identify integration sites of the transferred lentiviral vectors; 2) map the sites in the human genome and determine their relationship with structural and regulatory elements; 3) using quantitative chromatin profiling, test the DNAse I sensitivity of the integration sites and surrounding sequences, to obtain a direct in vivo assessment of whether lentiviral vectors integrate in open chromatin or in the proximity of activity regulatory elements.