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，并成为开发强直性肌营养不良新疗法的领导者。为了继续朝着这个目标前进，我建议探索纠正强直性1型肌营养不良症(DM1)诱导的多能干细胞(IPS)中突变基因的方法。我在医学硕士、博士生培训和博士后研究方面接受过科学研究方面的培训。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。我和我的导师制定了一份职业发展计划。我已经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极好的环境。我的短期目标是使用DM1iPS细胞作为平台来测试我们的假设，即DM1iPS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。我已经为这个目标开发了一个研究项目。DM1是由19号染色体上肌强直肌营养不良症蛋白激酶基因3‘-非翻译区的CTG核苷酸重复序列扩增引起的。该重复序列编码有毒的CUG RNA重复序列，隔离剪接因子并形成核内RNA焦点，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。患者特有的iPS细胞正在使这一点成为现实。然而，患者特有的iPS细胞仍然保留了致病突变，并可能在移植后经历相同的退化过程。为了利用这些细胞，突变的基因需要在移植前得到纠正。我们利用TALEN技术和同源重组(HR)设计了两种纠正突变基因的方法：1)靶向阻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中断转录的序列。2)定向删除扩展的重复序列。使用HR在扩展的重复序列之前和之后插入loxP位点。在Cre重组酶瞬时表达后，在loxP位点两侧的扩展重复序列将被删除。我们将通过类胚体介导的分化和畸胎瘤的形成，进一步评估基因组校正的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则性数据证据。这将克服自体细胞替代疗法发展中的一个障碍。建议项目的实施和K08机制下的培训将为我成功竞争R01拨款，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.