Gene Therapy for BETA ZERO Thalassemia

该项目的目标是进行临床前和临床研究，旨在测试造血干细胞动员和基因治疗在重型β-零地中海贫血中的安全性和有效性。零β地中海贫血，即完全缺乏β链合成的地中海贫血，已被选为靶疾病，因为尽管存在输血的红细胞，但仍有可能使用有效的定量细胞化学和生化技术来鉴定患者血液中新产生的β珠蛋白链和RNA。临床部 研究将在希腊进行，因为可以登记的贝塔0地中海贫血主要患者的数量很多。我们的具体目标是1)改进在当前资金周期中开发的β珠蛋白基因慢病毒载体。我们将完成已经启动的旨在生产β慢病毒载体的研究，该载体也携带绝缘体元件，有望减少位置效应并提高载体安全性。2)探讨干细胞动员治疗重型β-零地中海贫血的安全性和有效性。将使用三种协议。非脾切除组(6例)、非脾切除组(6例)和单纯G-CSF组(6例)。3)确定由HSC动员、马法兰调理和慢病毒载体转导的CD34+细胞自体移植组成的基因转移方案的安全性和有效性。12名患者将按剂量递增方案(70、100、120微克/平方米)接受美法兰治疗。四名患者将不接受调理，并作为对照组。4)检测转导的CD34+细胞输注后的β珠蛋白基因转移率和表达的持续性。移植后载体表达的分析将基于对A网织红细胞的定量，通过定量聚合酶链式反应测量血液中的β/α珠蛋白mRNA比率，以及分析红系祖细胞集落中的珠蛋白基因表达。5)研究转导慢病毒载体的整合生物学。使用基因组技术，我们将1)鉴定转移的慢病毒载体的整合位点；2)绘制人类基因组中的位点并确定它们与 结构和调控元件；3)使用定量染色质图谱，测试整合位点和周围序列的DNaseI敏感性，以获得体内直接评估慢病毒载体是整合在开放染色质中还是整合在活性调控元件附近。

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.