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

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肌强直性营养不良新疗法的领导者。为了继续实现这一目标，我建议探索纠正1型肌强直性营养不良（DM1）诱导的多能干细胞（iPS）突变基因的方法。在我的医学博士、博士生培养和博士后研究中，我接受了科学研究方面的训练。我相信K08导师职业发展奖将极大地促进我成为一名独立的研究科学家。我和导师一起制定了职业发展计划。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良好的环境。我的短期目标是使用DM1 iPS细胞作为平台来验证我们的假设，即DM1 iPS细胞中的突变基因可以使用转录激活物样效应核酸酶（TALEN）技术有效地纠正。为此，我开发了一个研究项目。DM1是由19号染色体上营养不良肌强直蛋白激酶基因3'-非翻译区CTG核苷酸重复扩增引起的。扩增的重复序列编码有毒的CUG RNA重复序列，这些重复序列隔离剪接因子并形成核内RNA灶，导致基因剪接异常和随后的临床表现。再生医学为晚期强直性肌营养不良的治疗带来了希望。针对患者的iPS细胞使这成为现实。然而，患者特异性iPS细胞仍然保留致病突变，并可能在移植后经历相同的退行性过程。为了利用这些细胞，需要在移植前纠正突变基因。我们利用TALEN技术和同源重组（homologous recombination， HR）设计了两种纠正突变基因的方法：1)靶向中断扩增重复序列的转录。在终止密码子和通过HR扩展的重复序列之间插入一个序列中断转录。2)扩增重复序列的靶向删除。使用HR在扩展重复之前和之后插入LoxP位点。在瞬时表达Cre-recombinase后，LoxP位点两侧的扩增重复序列将被删除。我们将进一步通过胚胎体介导的分化和畸胎瘤形成来评估基因组校正的DM1 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纠正。我们期望本研究能为TALEN技术用于纠正显性遗传突变基因提供原理数据证明。这将克服自体细胞替代疗法发展中的一个障碍。项目的实施和K08机制下的培训将为我成功竞争R01基金，成为一名独立的临床研究科学家做好准备。

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.