Genetic correction of human beta-thalassemic induced pluripotent stem cells

描述（由申请人提供）：本项目旨在为使用人类诱导多能干细胞（hiPSC）对（-地中海贫血）进行基因校正提供原理证明。hiPSC技术为基因修饰提供了独特的机会，从而改进和原则上更安全的基因治疗方法。与此同时，它带来了新的风险，需要在充分发挥其治疗潜力之前加以克服。我建议建立一个基于hipsc的模型，用于地中海贫血的基因校正。地中海贫血是一种由影响珠蛋白合成的突变引起的遗传性严重贫血，在地中海血统的受试者中非常突出。利用hiPSC技术提供的独特可能性，我将开发两种新的珠蛋白基因互补方法，旨在克服当前插入性肿瘤发生的风险：(a)选择“安全”的克隆，其中包含整合的慢病毒载体；(b)开发有丝分裂稳定的自我复制的片段载体。首先，我建议使用临床相关的方法从重度地中海贫血（thal-iPS）患者中生成hiPSCs，并制定优化的方案，使其分化为最终可移植的造血干细胞和红系祖细胞。其次，使用该疾病模型，我将通过慢病毒介导的正常（珠蛋白基因）等位基因的转移来纠正疾病，然后根据有利和“安全”的整合位点选择克隆。第三，我将开发具有编码珠蛋白基因的有丝分裂稳定性和染色体外复制能力的载体（由支架/基质附着区（S/MAR）赋予），并在thal-iPS模型中评估其长期持久性和治疗效果。第四，我将开发“failsafe”自杀基因策略，用于在移植前清除畸胎瘤起始细胞和移植后控制hipsc。

DESCRIPTION (provided by applicant): This project is intended to provide proof-of-principle for the genetic correction of (-thalassemia major using human induced pluripotent stem cells (hiPSC). hiPSC technology offers unique opportunities for genetic modification towards improved and, in principle, safer gene therapy approaches. At the same time, it imposes new risks that need to be overcome before its full therapeutic potential can be realized. I propose to establish a hiPSC-based model for genetic correction of (-thalassemia - an inherited severe form of anemia caused by mutations affecting ( globin synthesis - that is very prominent in subjects of Mediterranean origin. Taking into advantage the unique possibilities that hiPSC technology offers, I will develop two novel approaches of ( globin gene complementation aimed at overcoming current risks of insertional oncogenesis: (a) selection of "safe" clones harboring integrated lentiviral vectors and (b) development of mitotically stable self-replicating episomal vectors. First, I propose to generate hiPSCs from patients with (-thalassemia major (thal-iPS) using clinically relevant methodologies and to develop optimized protocols for their differentiation into definitive transplantable hematopoietic stem cells and erythroid progenitors. Second, using this disease model, I will correct the disease through lentiviral-mediated transfer of a normal (-globin gene allele followed by selection of clones on the basis of favorable and "safe" integration sites. Third, I will develop vectors with the ability of mitotic stability and extra-chromosomal replication (conferred by a Scaffold/Matrix Attachment Region, S/MAR) encoding the ( globin gene and assess their long-term persistence and therapeutic efficacy in the thal-iPS model. Fourth, I will develop "failsafe" suicide gene strategies for purging of teratoma-initiating cells before transplantation and control of hiPSCs after transplantation. The proposed studies will provide a new paradigm of gene therapy strategies based on hiPSCs with broader applications in the genetic correction of diseases of the hematopoietic system and will advance this newly emerging field towards translation to the clinic. I am currently a post-doctoral research fellow in the laboratory of Michel Sadelain at Memorial Sloan-Kettering Cancer Center in New York City. Dr Sadelain's laboratory has performed over the last decade pioneering research in the field of gene therapy of (-thalassemia. This project will facilitate my transition from mentored trainee to independent investigator. This laboratory at this institution offers an excellent environment to foster this transition. PUBLIC HEALTH RELEVANCE: With the recent advent of human induced pluripotent stem cell (hiPSC) technology, one can now use easily accessible somatic cells from adult individuals (e.g. from a skin biopsy or even a single hair) to create patient-specific pluripotent stem cells, differentiate them into the cell type needed and return the cells to the patient for therapeutic purposes. This project capitalizes on these recent advances in the generation of patient-specific hiPSCs and intends to combine this technology with novel genetic correction strategies. We will create a hiPSC-based model of (-thalassemia major, a severe form of anemia with high prevalence among individuals of Mediterranean origin, which constitutes the most common single gene disorder encountered in the human population. New gene therapy approaches, in principle safer and devoid of risks of insertional oncogenesis will be applied to cure this disease. This study will constitute a new paradigm for the treatment of genetic diseases, in general, as the genetic strategies that will be developed can potentially be applied to correct autologous iPSCs from patients with any genetic disorder. It will also advance the field of pluripotent stem cell transplantation therapies towards the clinic. This field is posed to find broad applications, as many diseases currently treated by allogeneic BMT are amenable to transplantation of autologous HSCs derived in vitro from iPSCs.