Genetic correction of human beta-thalassemic induced pluripotent stem cells

简介（由申请人提供）：候选人：本人在希腊获得医学博士和博士学位。在过去的四年里，我一直在纽约纪念斯隆-凯特琳癌症中心的Michel Sadelain博士的实验室做博士后，在那里我通过利用microrna介导的基因调控，研究造血系统中慢病毒编码的转基因的谱系和发育阶段特异性表达的工程，以及患者特异性诱导多能干细胞（iPSCs）的产生和遗传修饰。我的长期目标是开发更安全的基因工程方法来治疗血液疾病。获得NIH独立之路奖（K99/R00）将使我能够在奖项的指导阶段获得额外的培训，包括研讨会、课程、科学会议、指导技能的发展和研究技术的培训，如人类基因组的生物信息学分析和人类ipcs的造血分化。通过额外的培训，我将能够在一个高排名的学术研究机构从事独立的研究工作，并将我的职业生涯集中在转化干细胞研究上。环境：纪念斯隆-凯特琳癌症中心（MSKCC）是一个生物医学研究中心，汇集了科学家和医生，共同致力于将基础科学转化为临床前和临床研究。这种环境强烈鼓励跨学科和合作的调查项目，并提供许多培训和教育机会。此外，三机构协作网络是由MSKCC，洛克菲勒大学和威尔康奈尔医学院组成的联合倡议，支持更广泛的网络机会以及跨机构共享核心设施资源。Sadelain博士的实验室是细胞工程中心（CCE）的一部分，该中心汇集了来自干细胞生物学、基因工程、自体细胞传递和转基因调控等领域的研究人员。研究：为了实现诱导多能干细胞（iPSCs）在再生医学中的应用前景，需要对其进行精确、安全的基因修饰和清除残留的分化抗性细胞的策略。这项K99/R00申请的目的是开发和评估一种基因添加策略，用于使用患者特异性iPSCs治疗常见遗传性血液疾病ss-地中海贫血的自体细胞治疗。该方法利用多能干细胞的基因工程，将疾病纠正与对未分化细胞的保护结合起来，旨在规避因未分化多能干细胞的随机整合和持续存在而带来的肿瘤发生风险。具体目标是：(1)生成含有慢病毒编码？-珠蛋白转基因整合在“安全港”基因组位点。无转基因的半诱导多能干细胞将用编码ss-珠蛋白的慢病毒载体和可交换的Neo-eGFP选择盒进行转导。单载体复制整合子将根据抗沉默转基因表达和载体染色体位置进行筛选，“安全港”整合位点将根据与内源性基因，特别是与癌症相关基因的接近程度进行选择。(2)设计一种“自杀基因”策略来清除具有分化抗性的thal-iPSCs。单纯疱疹病毒胸苷激酶（HSV-tk）“自杀基因”通过组织特异性启动子/增强子和/或miR- NAs调控表达，选择性地消除未分化的thal-iPSC，而不是其分化的后代，将通过重组酶介导的盒式交换（RMCE）工程设计并纳入预先选择的thal-iPSC克隆。(3)描述a ？-球蛋白和一个HSV-tk基因在半多能干细胞的“安全港”位点整合。在“安全港”位点整合的ss-球蛋白和HSV-tk转基因的组织特异性和表达水平，以及邻近基因的表达，将在未分化的半ipsc克隆及其红系后代中确定。更昔洛韦给药后未分化肿瘤起始细胞的清除将在体外和畸胎瘤形成试验中进行评估。本研究利用生物信息学分析和iPSCs及其分化后代的基因表达谱，提出了一种定义和框架，用于对人类基因组中转基因整合的“安全港”位点进行前瞻性鉴定。该项目还利用转录后调控的新机制，通过利用发育过程中不同的microRNA表达模式来设计转基因表达的稳健控制。清除未分化细胞的“自杀基因”策略可以广泛适用于再生医学中所有基于多能干细胞的治疗。在人类多能干细胞技术的新时代，这项原理验证研究可以提供基于ips的细胞和基因综合治疗的新范例，普遍适用于遗传疾病，并推动这一新领域向临床转化。

DESCRIPTION (provided by applicant): Candidate: I obtained my M.D. and Ph.D. in Greece. For the past four years I have been a postdoctoral fellow in the laboratory of Dr Michel Sadelain at Memorial Sloan-Kettering Cancer Center in New York, where I worked on the engineering of lineage- and developmental stage- specific expression of lentivirally-encoded transgenes in the hematopoietic system by exploiting microRNA-mediated gene regulation and on the generation and genetic modification of patient-specific induced pluripotent stem cells (iPSCs). My long-term goal is to develop safer genetic engineering approaches for the treatment of blood disorders. Obtaining an NIH Pathway to Independence Award (K99/R00) will allow me to gain additional training in the mentored phase of the award with activities such as seminars, courses, scientific conferences, development of mentoring skills and training in research techniques such as bioinformatics analyses of the human genome and hematopoietic differentiation of human iPCSs. With additional training, I will be able to pursue an independent research position in a highly ranked academic research institution and focus my career in translational stem cell research. Environment: Memorial Sloan-Kettering Cancer Center (MSKCC) is a center of biomedical research bringing together scientists and physicians working together towards translation of basic science to preclinical and clinical research. This environment strongly encourages interdisciplinary and collaborative investigative projects and offers many training and educational opportunities. Additionally, the Tri-Institutional Collaboration Network is a joint initiative comprising MSKCC, The Rockefeller University, and Weill Cornell Medical College and supports broader networking opportunities as well as sharing of core facility resources across institutions. Dr Sadelain's laboratory is part of the Center for Cell Engineering (CCE), which brings together researchers from areas that encompass stem cell biology, genetic engineering, autologous cell delivery and transgene regulation. Research: For the promise of induced pluripotent stem cells (iPSCs) in regenerative medicine to be realized, strategies for their precise and safe genetic modification and for purging of residual differentiation-resistant cells are needed. The objective of this K99/R00 application is to develop and evaluate a gene addition strategy for autologous cell therapy of a common inherited blood disorder, ss-thalassemia major, using patient-specific iPSCs. The approach uses genetic engineering of iPSCs, integrating disease correction with protection against undifferentiated cells, with the aim to circumvent risks of oncogenesis posed by both random integration and persistence of undifferentiated pluripotent stem cells. The specific aims are: (1) To generate ss-thalassemia iPSCs (thal-iPSCs) harboring a lentivirally-encoded ?-globin transgene integrated at "safe harbor" genomic sites. Transgene-free thal-iPSCs will be transduced with a lentiviral vector encoding ss-globin and an ex- changeable Neo-eGFP selection cassette. Single vector copy integrants will be screened according to silencing-resistant transgene expression and vector chromosomal position and "safe harbor" integration sites will be selected, based on proximity to endogenous genes, especially cancer-related genes. (2) To engineer a "sui- cide gene" strategy for purging of differentiation-resistant thal-iPSCs. An Herpes Simplex Virus-thymidine kinase (HSV-tk) "suicide gene" with regulated expression by tissue-specific promoters/enhancers and/or miR- NAs to selectively eliminate undifferentiated thal-iPSCs but not their differentiated progeny will be engineered and incorporated in pre-selected thal-iPSC clones through recombinase-mediated cassette exchange (RMCE). (3) To characterize the therapeutic and safety features conferred by a ?-globin and an HSV-tk transgene inte- grated at "safe harbor" sites in thal-iPSCs. The tissue specificity and levels of expression of the ss-globin and HSV-tk transgenes integrated at "safe harbor" sites, as well as the expression of neighboring genes, will be determined in undifferentiated thal-iPSC clones and their erythroid progeny. Purging of undifferentiated tumor- initiating cells after administration of ganciclovir will be assessed in vitro and in teratoma formation assays. This study proposes a definition and framework for the prospective identification of "safe harbor" sites for transgene integration in the human genome, using bioinformatics analyses and gene expression profiling of iPSCs and their differentiated progeny. This project also harnesses novel mechanisms of post-transcriptional regulation to engineer robust control of transgene expression by exploiting distinct microRNA expression pat- terns during development. The "suicide gene" strategy for purging of undifferentiated cells can be broadly applicable to all pluripotent stem cell-based therapies in regenerative medicine. In the new era of human pluripotent stem cell technology, this proof-of-principle study can provide a new paradigm of integrated iPS-based cell and gene therapy, generally applicable to genetic disorders and advance this new field towards translation to the clinic.