Genetic correction of human beta-thalassemic induced pluripotent stem cells

描述(由申请人提供)：该项目旨在为使用人类诱导多能干细胞(HiPSC)对(-地中海贫血)进行基因矫正提供原则证明。HiPSC技术为基因改造提供了独特的机会，从而改进了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，需要在充分实现其治疗潜力之前克服这些风险。我建议建立一个基于hiPSC的模型，用于地中海贫血的遗传纠正-地中海贫血是一种遗传性严重形式的贫血，由影响珠蛋白合成的突变引起，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，旨在克服目前插入癌形成的风险：(A)选择含有整合慢病毒载体的“安全”克隆；(B)开发有丝分裂稳定的自我复制的上体载体。首先，我建议使用临床相关方法从重型地中海贫血(Thal-iPS)患者中培养hPSCs，并开发优化的方案，使其分化为最终可移植的造血干细胞和红系祖细胞。其次，使用这种疾病模型，我将通过慢病毒介导的正常(-珠蛋白)基因等位基因的转移，然后在有利和“安全”整合位点的基础上选择克隆来纠正疾病。第三，我将开发编码珠蛋白基因的具有有丝分裂稳定和染色体外复制能力的载体(由支架/基质附着区S/MAR授予)，并评估其在Thal-iPS模型中的长期持久性和治疗效果。第四，我将开发“失败安全”的自杀基因策略，在移植前清除启动畸胎瘤的细胞，并在移植后控制HiPSCs。 这些研究将为基于HiPSCs的基因治疗策略提供一个新的范例，在造血系统疾病的基因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·萨德兰实验室的博士后研究员。在过去的十年里，萨德兰博士的实验室在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转变。该机构的实验室为促进这一过渡提供了良好的环境。 公共卫生相关性：随着最近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人个体的容易获得的体细胞(例如，从皮肤活检或甚至从一根头发中)来创建针对患者的多能干细胞，将它们分化为所需的细胞类型，并将这些细胞返回给患者用于治疗目的。该项目利用这些最新的进展来产生针对患者的HiPSCs，并打算将这项技术与新的基因纠正策略相结合。我们将创建一个基于HiPSC的重型地中海贫血模型，这是一种严重的贫血形式，在地中海血统的个人中发病率很高，构成了人类人口中遇到的最常见的单基因疾病。新的基因治疗方法，原则上更安全，没有插入致癌的风险，将被应用于治愈这种疾病。这项研究将构成治疗遗传性疾病的新范式，因为将开发的遗传策略可能被应用于纠正任何遗传性疾病患者的自体IPSCs。这也将推动多能干细胞移植治疗领域走向临床。这一领域具有广阔的应用前景，因为目前用异基因骨髓移植治疗的许多疾病都可以移植从IPSCs体外获得的自体HSCs。

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.