Genetic correction of human beta-thalassemic induced pluripotent stem cells

人β地中海贫血诱导多能干细胞的遗传校正

• 批准号: 8955926
• 负责人: Eirini Papapetrou
• 金额: $ 14.41万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8955926
• 关键词: Area; Autologous; Award; Basic Science; Bioinformatics; Biological Assay; Biomedical Research; Cell Line; Cell Therapy; Cells; Clinic; Clinical Research; Collaborations; Core Facility; Development; Disease; Distant; Doctor of Medicine; Doctor of Philosophy; Engineering; Enhancers; Environment; Erythroid; Foundations; Ganciclovir; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Engineering; Genome; Genomics; Globin; Goals; Greece; HSV-Tk Gene; Hematological Disease; Hematopoietic; Hematopoietic System; Hereditary Disease; Human; Human Genome; Immunodeficient Mouse; In Vitro; Inherited; Institution; Joints; Laboratories; Lead; Lentivirus Vector; Malignant Neoplasms; Mediating; Memorial Sloan-Kettering Cancer Center; Mentors; Methodology; MicroRNAs; Microarray Analysis; Modification; New York; Oncogenic; Pathway interactions; Patients; Pattern; Phase; Physicians; Pluripotent Stem Cells; Positioning Attribute; Post-Transcriptional Regulation; Postdoctoral Fellow; Regenerative Medicine; Regulation; Research; Research Personnel; Research Technics; Residual state; Resistance; Resources; Risk; Safety; Scientist; Simplexvirus; Site; Specificity; Staging; Stem Cell Research; Stem cells; Teratoma; Therapeutic; Thymidine Kinase; Time; Tissues; Training; Transgenes; Translations; Undifferentiated; United States National Institutes of Health; Universities; Work; base; career; cellular engineering; erythroid differentiation; expression vector; frontier; gene therapy; induced pluripotent stem cell; medical schools; novel; pre-clinical research; promoter; prospective; purge; recombinase; skills training; stem cell biology; stem cell technology; suicide gene; symposium; transgene expression; tumor; tumorigenesis; vector

PROJECT SUMMARY/ABSTRACT 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, beta halassemia 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 beta halassemia iPSCs (thal-iPSCs) harboring a lentivirally-encoded ss-globin transgene integrated at "safe harbor" genomic sites. Transgene-free thal-iPSCs will be transduced with a lentiviral vector encoding ss-globin and an exchangeable 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 "suicide 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 ss-globin and an HSV-tk transgene integrated 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 patterns 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.

项目摘要/摘要 应聘者：我在希腊获得医学博士和博士学位。在过去的四年里，我一直是中国的博士后研究员 米歇尔·萨德兰博士在纽约纪念斯隆-凯特琳癌症中心的实验室，我在那里工作 慢病毒编码的转基因的谱系和发育阶段特异性表达工程 利用microRNA介导的基因调控的造血系统及其发生和遗传 患者特异性诱导多能干细胞(IPSCs)的修饰。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。获得美国国立卫生研究院独立之路 奖项(K99/R00)将允许我在奖项的指导阶段通过活动获得额外的培训 例如研讨会、课程、科学会议、指导技能发展和研究培训 人类基因组生物信息学分析和人类造血分化等技术 IPCSS。通过额外的培训，我将能够在一家排名很高的学术研究机构寻求一个独立的研究职位，并将我的职业生涯集中在翻译干细胞研究上。 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， 科学家和医生共同努力，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协作的调查项目 并提供许多培训和教育机会。此外，三个机构的合作网络 是一项由MSKCC、洛克菲勒大学和威尔·康奈尔医学院组成的联合计划，支持更广泛的网络机会以及各机构之间的核心设施资源共享。萨德兰博士的实验室是细胞工程中心(CCE)的一部分，该中心汇集了来自不同领域的研究人员 包括干细胞生物学、基因工程、自体细胞输送和转基因调控。 研究：为了实现再生医学中诱导多能干细胞(IPSCs)的前景， 其精确和安全的基因修饰和清除残余分化抗性的策略 细胞是必需的。该K99/R00应用的目的是开发和评估基因添加策略 用于一种常见遗传性血液疾病的自体细胞治疗，主要是贝塔哈贫血，使用患者特有的 IPSCs。这种方法使用了ipscs的基因工程，将疾病纠正与预防相结合。 未分化的细胞，目的是规避随机整合和 未分化的多能干细胞的持久性。具体目标是：(1)产生β-半贫血 携带慢病毒编码的ss-珠蛋白转基因的IPSCs(Thal-IPSCs)整合在“安全港”基因组中 网站。无转基因的Thal-iPSCs将通过编码ss-珠蛋白的慢病毒载体和可交换的Neo-EGFP选择盒进行转导。将根据抗沉默转基因表达和载体染色体位置筛选单载体拷贝整合子，并将整合部位设为安全港 根据与内源性基因，特别是与癌症相关的基因的接近程度进行选择。(2)设计一种“自杀基因”策略，清除具有分化抗性的Thal-iPSCs。一种单纯疱疹病毒胸腺嘧啶核苷 受组织特异性启动子/增强子和/或miR调控表达的自杀基因 选择性地消除未分化的Thal-iPSCs但不是其分化的后代的NAS将被设计出来 并通过重组酶介导的盒式交换(RMCE)整合到预选的Thal-IPSC克隆中。 (3)研究整合在Thal-iPSCs“安全港”部位的单链珠蛋白和HSV-tk转基因的治疗和安全性。单链珠蛋白和单链珠蛋白的组织特异性和表达水平 整合在“安全港”的HSV-tk转基因，以及邻近基因的表达，将被 在未分化的Thal-IPSC克隆及其红系后代中确定。清除未分化的肿瘤- 更昔洛韦给药后的启动细胞将在体外和畸胎瘤形成试验中进行评估。 本研究为“安全港”地点的前瞻性识别提出了一个定义和框架。 利用生物信息学分析和基因表达谱分析在人类基因组中整合转基因 IPSCs及其分化的后代。该项目还利用了转录后转录后的新机制 通过在发育过程中利用不同的microRNA表达模式来设计对转基因表达的稳健控制的调控。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干细胞的多能疗法。在人类多能干细胞技术的新时代，这一原则验证研究可以提供一种基于iPS的整合细胞的新范式 和基因疗法，普遍适用于遗传性疾病，并推动这一新领域向 诊所。