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: 8588316
• 负责人: Eirini Papapetrou
• 金额: $ 11.77万
• 依托单位: UNIVERSITY OF WASHINGTON
• 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 2014-04-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8588316
• 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 候选人：我获得了我的医学博士学位和博士学位。在希腊。在过去的四年中，我一直是博士后研究员 纽约纪念斯隆 - 凯特林癌症中心的米歇尔·萨德兰（Michel Sadelain）博士的实验室，我在那里工作 谱系和发育阶段特异性表达的慢病毒编码转基因的工程 造血系统通过利用microRNA介导的基因调节以及产生和遗传 修饰患者特异性诱导的多能干细胞（IPSC）。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独立途径 奖项（K99/R00）将使我能够在奖项的指导阶段获得其他培训 例如研讨会，课程，科学会议，发展技能的发展和研究培训 人类基因组的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，我将能够在高级的学术研究机构中担任独立的研究职位，并将我的职业重点放在翻译干细胞研究中。 环境：纪念斯隆 - 凯特林癌症中心（MSKCC）是生物医学研究的中心 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，三机构合作网络 是由MSKCC，Rockefeller大学和Weill Cornell医学院组成的联合倡议，并支持更广泛的网络机会以及在机构之间共享核心设施资源。 Sadelain博士的实验室是细胞工程中心（CCE）的一部分，该中心将研究人员汇集在一起 涵盖了干细胞生物学，基因工程，自体细胞输送和转基因调节。 研究：为了实现再生医学中诱导多能干细胞（IPSC）的承诺， 其精确且安全的遗传修饰的策略以及抗剩余分化的策略 需要细胞。该K99/R00应用的目的是制定和评估基因添加策略 用于常见遗传性血液疾病的自体细胞疗法，使用患者特异 ipscs。该方法使用IPSC的基因工程，将疾病纠正与防御的保护相结合 未分化的细胞，目的是规避随机整合和 未分化多能干细胞的持久性。具体目的是：（1）产生βhalassemia IPSCS（thal-ipscs）携带慢病毒编码的SS-珠蛋白转基因在“安全港”基因组中集成 站点。无基因thal-ipsc将用编码SS-珠蛋白和可交换的Neo-EGFP选择盒的慢病毒载体转导。单矢量副本整合体将根据抗沉默的转基因表达和矢量染色体位置以及“安全港”整合位点进行筛选 基于与内源基因的接近性，尤其是与癌症相关的基因。 （2）设计一种“自杀基因”策略，用于清除耐药性thal-ipscs。单纯疱疹病毒 - 胸腺苷 激酶（HSV-TK）“自杀基因”，由组织特异性启动子/增强子和/或miR-- NAS可以选择性地消除未分化的thal-ipsc，但不会进行分化的后代 并通过重组酶介导的盒式磁带交换（RMCE）掺入预选的Thal-IPSC克隆中。 （3）表征由thal-ipscs的“安全港”地点集成的SS-球蛋白和HSV-TK Transgene赋予的治疗和安全特征。 SS-球蛋白的组织特异性和表达水平和 集成在“安全港”站点的HSV-TK转基因以及相邻基因的表达将是 在未分化的thal-ipsc克隆及其红细胞后代确定。清除未分化的肿瘤 给予Ganciclovir后的细胞将在体外和畸胎瘤形成测定中进行评估。 这项研究提出了一个定义和框架，以确定“安全港”站点 用于人类基因组中的转基因整合，使用生物信息学分析和基因表达分析 IPSC及其差异化后代。该项目还利用了转录后的新型机制 通过利用开发过程中利用不同的microRNA表达模式来调节对转基因表达的强大控制。清除未分化细胞的“自杀基因”策略可以广泛适用于所有多能干细胞的再生医学疗法。在人类多能干细胞技术的新时代，这项原理研究可以提供基于IPS的综合细胞的新范式 和基因疗法，通常适用于遗传疾病，并将这一新领域推向转化为 诊所。