Genome correction of Myotonic Dystrophy type 1 iPS cells by TALEN technology

通过TALEN技术对强直性肌营养不良1型iPS细胞进行基因组校正

• 批准号: 9538346
• 负责人: Guangbin Xia
• 金额: $ 5.36万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9538346
• 关键词: Genome; correction; Myotonic; Dystrophy; type

DESCRIPTION (provided by applicant): My long career goal is to become an independent clinical research scientist and a leader in developing novel therapies for myotonic dystrophy. To continue my progress towards this goal, I propose to explore approaches to correct the mutant gene in Myotonic Dystrophy type 1 (DM1) induced pluripotent stem (iPS) cells. I have been trained in conducting scientific research in my MD, PhD student training and postdoctoral studies. I believe the K08 mentored career development award will greatly facilitate me to become an independent research scientist. I have developed a career development plan with my mentor. I have assembled a strong mentor committee. University of Florida, McKnight Brain Institute, Department of Neuroscience and Neurology, Center for Cellular Reprogramming and Center for NeuroGenetics provide an excellent environment for me to pursue the long term goal. My short-term goal is to use DM1 iPS cells as a platform to test our hypothesis that mutant gene in DM1 iPS cells can be efficiently corrected using transcription activator-like effector nuclease (TALEN) technology. I have developed a research project for this goal. DM1 is caused by CTG nucleotide repeat expansion within the dystrophia myotonica protein kinase gene 3'-untranslated region on chromosome 19. The expanded repeats encode toxic CUG RNA repeats, which sequester splicing factors and form intranuclear RNA foci, leading to aberrant gene splicing and subsequent clinical manifestations. Regenerative medicine holds hope for treatment of advanced myotonic dystrophy. Patient-specific iPS cells are making this realistic. However, patient-specific iPS cells still retain the pathogenic mutation and may undergo the same degenerative process after transplantation. To utilize these cells, the mutated gene needs to be corrected before transplantation. We have designed two approaches in correcting the mutant gene using TALEN technology and homologous recombination (HR): 1) targeted interruption of transcription of the expanded repeats. A sequence interrupting transcription will be inserted between the stop codon and expanded repeats through HR. 2) targeted deletion of the expanded repeats. LoxP sites will be inserted before and after the expanded repeat using HR. The expanded repeats flanked by LoxP sites will then be deleted following transient expression of Cre-recombinase. We will further evaluate in vitro and in vivo pluripotency of genome- corrected DM1 iPS cells by embryoid body-mediated differentiation and teratoma formation. We will evaluate RNA foci and downstream splicing events to confirm the successful correction of molecular phenotypes. We expect this study will provide proof of principle data that TALEN technology can be used to correct dominantly inherited mutant genes. This will overcome one of the hurdles in the development of autologous cell replacement therapy. The implementation of the proposed project and training under K08 mechanism will well- prepare me to compete successfully for R01 funding to become an independent clinical research scientist.

描述（由申请人提供）：我的长期职业目标是成为一名独立的临床研究科学家和开发强直性肌营养不良症新疗法的领导者。为了继续实现这一目标，我建议探索纠正强直性肌营养不良1型（DM1）诱导的多能干细胞（iPS）中突变基因的方法。我在我的MD，博士生培训和博士后研究中接受了科学研究的培训。我相信K08指导职业发展奖将大大促进我成为一个独立的研究科学家。我和我的导师制定了一个职业发展计划。我组建了一个强大的导师委员会。佛罗里达大学、麦克奈特脑研究所、神经科学和神经病学系、细胞重编程中心和神经遗传学中心为我追求长期目标提供了一个很好的环境。我的短期目标是使用DM1 iPS细胞作为平台来测试我们的假设，即DM1 iPS细胞中的突变基因可以使用转录激活因子样效应核酸酶（TALEN）技术有效地纠正。我已经为这个目标开发了一个研究项目。DM 1是由19号染色体上肌强直性营养不良蛋白激酶基因3 '非翻译区内的CTG核苷酸重复扩增引起的。扩增的重复序列编码毒性CUG RNA重复序列，其隔离剪接因子并形成核内RNA灶，导致异常基因剪接和随后的临床表现。再生医学为治疗晚期强直性肌营养不良带来希望。患者特异性iPS细胞正在使这成为现实。然而，患者特异性iPS细胞仍然保留致病突变，并且在移植后可能经历相同的退化过程。为了利用这些细胞，需要在移植前纠正突变的基因。我们设计了两种利用TALEN技术和同源重组（HR）纠正突变基因的方法：1）靶向中断扩增重复序列的转录。通过HR将中断转录的序列插入终止密码子和扩增重复序列之间。2）扩增重复序列的靶向缺失。使用HR将LoxP位点插入扩增的重复序列之前和之后，然后在Cre重组酶的瞬时表达后将侧翼为LoxP位点的扩增的重复序列删除。我们将通过胚状体介导的分化和畸胎瘤形成进一步评估基因组校正的DMl iPS细胞的体外和体内多能性。我们将评估RNA焦点和下游剪接事件，以确认分子表型的成功校正。我们希望这项研究将提供TALEN技术可用于纠正显性遗传突变基因的原理数据证明。这将克服自体细胞替代疗法发展中的一个障碍。拟议项目的实施和K08机制下的培训将使我做好充分准备，成功竞争R01资金，成为一名独立的临床研究科学家。