Genetic correction of human beta-thalassemic induced pluripotent stem cells

描述（由申请人提供）：候选人：我获得了医学博士学位。和博士在希腊。在过去的四年里，我一直在纽约纪念斯隆-凯特琳癌症中心的米歇尔·萨德莱恩博士的实验室做博士后研究员，在那里，我致力于通过利用microRNA介导的基因调控，在造血系统中对慢病毒编码的转基因进行谱系和发育阶段特异性表达的工程设计，以及对患者特异性诱导多能干细胞（iPSC）的生成和遗传修饰。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/R 00）将使我能够在该奖项的指导阶段获得额外的培训，如研讨会，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。工作环境：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。这种环境大力鼓励跨学科和合作调查项目，并提供许多培训和教育机会。此外，三机构合作网络是一个联合倡议，包括MSKCC，洛克菲勒大学和威尔康奈尔医学院，并支持更广泛的网络机会，以及跨机构的核心设施资源共享。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、自体细胞输送和转基因调控等领域的研究人员。调研：为了实现诱导多能干细胞（iPSC）在再生医学中的前景，需要对其进行精确和安全的遗传修饰以及清除残留的抗分化细胞的策略。该K99/R 00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iPSC对常见遗传性血液疾病重型β地中海贫血进行自体细胞治疗。该方法使用iPSC的基因工程，将疾病矫正与对未分化细胞的保护相结合，旨在规避由未分化多能干细胞的随机整合和持续存在所带来的肿瘤发生风险。具体目标是：（1）产生具有慢病毒编码的β-地中海贫血iPSCs（thal-iPSCs），在“安全港”基因组位点整合的球蛋白转基因。将用编码β-珠蛋白的慢病毒载体和可交换的Neo-eGFP选择盒转导无转基因的thal-iPSC。将根据沉默抗性转基因表达和载体染色体位置筛选单载体拷贝整合体，并基于与内源基因，特别是癌症相关基因的接近度选择“安全港”整合位点。(2)设计“锡德基因”策略以清除抗分化的thal-iPSC。通过组织特异性启动子/增强子和/或miR-NA调节表达以选择性消除未分化的thal-iPSC但不消除其分化的后代的单纯疱疹病毒-胸苷激酶（HSV-tk）“自杀基因”将被工程化并通过重组酶介导的盒交换（RMCE）掺入预选的thal-iPSC克隆中。(3)描述？-珠蛋白和HSV-tk转基因在thal-iPSC中的“安全港”位点整合。将在未分化的thal-iPSC克隆及其红系后代中测定整合在“安全港”位点的β-珠蛋白和HSV-tk转基因的组织特异性和表达水平以及相邻基因的表达。将在体外和畸胎瘤形成试验中评估更昔洛韦给药后未分化肿瘤起始细胞的清除。 这项研究提出了一个定义和框架的前瞻性识别的“安全港”网站的转基因整合在人类基因组中，使用生物信息学分析和基因表达谱的iPSCs及其分化的后代。该项目还利用转录后调控的新机制，通过利用发育期间不同的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疗法。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一种新的基于iPS的综合细胞和基因治疗的范例，通常适用于遗传疾病，并将这一新领域推向临床。

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.