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诱导多能干细胞（hiPSC）进行重型β地中海贫血的遗传校正提供原理证明。hiPSC技术为基因修饰提供了独特的机会，以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，在实现其全部治疗潜力之前需要克服这些风险。我建议建立一个基于hiPSC的模型，用于遗传校正β-地中海贫血-一种遗传性严重贫血，由影响β-珠蛋白合成的突变引起-这在地中海起源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，旨在克服目前插入肿瘤发生的风险：（a）选择携带整合慢病毒载体的“安全”克隆和（B）开发有丝分裂稳定的自我复制附加型载体。首先，我建议使用临床相关的方法从患有α-地中海贫血（Thal-iPS）的患者中产生hiPSC，并开发优化的方案，使其分化为永久性可移植造血干细胞和红系祖细胞。其次，使用这种疾病模型，我将通过慢病毒介导的正常β-珠蛋白基因等位基因转移，然后根据有利和“安全”的整合位点选择克隆来纠正疾病。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（由支架/基质附着区（S/MAR）赋予）的载体，并评估它们在thal-iPS模型中的长期持久性和治疗功效。第四，我将开发“故障安全”自杀基因策略，用于在移植前清除畸胎瘤起始细胞和在移植后控制hiPSC。 拟议的研究将提供基于hiPSC的基因治疗策略的新范例，在造血系统疾病的遗传矫正中具有更广泛的应用，并将推动这一新兴领域向临床转化。 我目前是纽约市纪念斯隆-凯特琳癌症中心米歇尔·萨德莱恩实验室的博士后研究员。Sadelain博士的实验室在过去十年中在β-地中海贫血的基因治疗领域进行了开创性的研究。这个项目将促进我从指导实习生到独立调查员的过渡。这个实验室在这个机构提供了一个很好的环境，以促进这种转变。 公共卫生相关性：随着人类诱导多能干细胞（hiPSC）技术的出现，人们现在可以使用来自成年个体（例如来自皮肤活检或甚至单根头发）的易于获得的体细胞来创建患者特异性多能干细胞，将它们分化为所需的细胞类型并将细胞返回患者用于治疗目的。该项目利用了这些在产生患者特异性hiPSC方面的最新进展，并打算将该技术与新型遗传校正策略联合收割机相结合。我们将建立一个基于hiPSC的重型地中海贫血模型，这是一种严重的贫血，在地中海血统的个体中患病率很高，构成了人群中最常见的单基因疾病。新的基因治疗方法，原则上更安全，没有插入肿瘤发生的风险将被应用于治疗这种疾病。这项研究将为遗传疾病的治疗提供一个新的范例，因为将开发的遗传策略可能适用于纠正任何遗传疾病患者的自体iPSC。它还将推动多能干细胞移植治疗领域走向临床。该领域被提出来寻找广泛的应用，因为目前通过同种异体BMT治疗的许多疾病适合于移植体外衍生自iPSC的自体HSC。

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.