Pathogenesis and CRISPR/Cas9 Correction of TCF4 Expansion in Fuchs Dystrophy

Fuchs 营养不良症中 TCF4 扩增的发病机制和 CRISPR/Cas9 校正

• 批准号: 9018954
• 负责人: ALBERT S JUN
• 金额: $ 24.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9018954
• 关键词: Accounting; Affect; Antisense RNA; Binding; Blindness; Cell Death; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cornea; Corneal Diseases; Corneal Endothelium; Corneal edema; Defect; Disease; Disease Clusterings; Endothelial Cells; Endothelium; Fluorescent in Situ Hybridization; Fuchs' Endothelial Dystrophy; Functional disorder; Future; Genes; Genetic Transcription; Genome; Genome engineering; Genomic DNA; Genomics; Goals; Guide RNA; Human; Immunohistochemistry; Keratoplasty; Label; Lead; Length; Mission; Modeling; Molecular; Mutation; Nonhomologous DNA End Joining; Oligonucleotides; Operative Surgical Procedures; Pathogenesis; Patients; Plasmids; Platelet Factor 4; Population; Protein Splicing; Proteins; Public Health; RNA; RNA Processing; Research; Role; Side; Surface; System; Testing; Therapeutic Human Experimentation; Tissues; Toxic effect; Translations; Trinucleotide Repeat Expansion; Trinucleotide Repeats; United States National Institutes of Health; Vision; Western Blotting; arm; base; design; endonuclease; gene correction; gene therapy; improved; nuclease; polyclonal antibody; public health relevance; repaired; transcription factor

 DESCRIPTION (provided by applicant): Fuchs endothelial corneal dystrophy (FECD) is characterized by endothelial cell death, corneal edema, and vision loss. FECD affects approximately 4% of the US population. The only definitive treatment is endothelial keratoplasty, and FECD was the leading indication for corneal transplant in the US in 2013. FECD is a corneal disease with major public health impact in the US and thus is relevant to the mission of the National Institutes of Health. A major unmet need for this important corneal disorder is better understanding of pathophysiology and improved, non-surgical treatments. Approximately 70% of FECD cases are caused by a trinucleotide repeat (TNR) expansion in the TCF4 gene. Major pathogenic mechanisms of TNR expansions involve 1) RNA toxicity from antisense transcription and 2) protein toxicity from repeat associated non-ATG (RAN) translation. Both of these mechanisms remain uncharacterized for the TCF4 mutation, and successful completion of this project would substantially expand our understanding of TNR associated pathogenesis of FECD. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) 9 nuclease are an extremely versatile and accurate approach to cut genomic DNA in numerous experimental systems. CRISPR/Cas9 have been used to delete and replace genomic sequence using non-homologous end joining (NHEJ) or homology directed repair (HDR). Use of this system for correction of the TCF4 TNR repeat in FECD has not been investigated and could lead to new treatment approaches. Overall goals of this project are to assess 1) the roles of antisense RNAs and RAN translation as pathogenic mechanisms and 2) the potential for CRISPR/Cas9 to correct the TCF4 TNR expansion. Aim I will test the hypothesis that the TCF4 TNR expansion: 1) produces toxic antisense RNAs which bind the RNA processing protein, muscleblind-like 1 (MBNL1) and 2) produces aberrant proteins resulting from repeat associated non-ATG (RAN) translation. Toxic antisense RNAs and MBNL1 binding will be identified by assessing for colocalization of RNA foci using labeled oligonucleotides and MBNL1 using fluorescence in situ hybridization. RAN translation products will be assessed by Western blotting of transformed human corneal endothelial cells transfected with plasmids encoding predicted RAN proteins and immunohistochemistry of FECD patient endothelial tissue using polyclonal antibodies raised against predicted RAN proteins. Aim II will test the hypothesis that CRISPR/Cas9 can be used to correct the TCF4 TNR expansion. Guide RNAs (gRNAs) will be designed on either side of the TCF4 TNR and will be assessed singly for cutting activity in 293 cells. The best gRNAs will be singly or doubly transfected with flanking oligonucleotides to assess for HDR, and doubly transfected without an oligonucleotide to assess for NHEJ in 293 cells and subsequently in transformed FECD corneal endothelial cells with the TCF4 TNR expansion.

 描述（由申请方提供）：Fuchs内皮角膜营养不良（FECD）的特征为内皮细胞死亡、角膜水肿和视力丧失。FECD影响约4%的美国人口。唯一确定的治疗方法是内皮角膜移植术，FECD是2013年美国角膜移植的主要适应症。FECD是一种在美国具有重大公共卫生影响的角膜疾病，因此与美国国立卫生研究院的使命相关。这种重要的角膜疾病的一个主要未满足的需求是更好地 了解病理生理学和改进的非手术治疗。 大约70%的FECD病例是由TCF 4基因中的三核苷酸重复（TNR）扩增引起的。TNR扩增的主要致病机制涉及1）来自反义转录的RNA毒性和2）来自重复相关非ATG（RAN）翻译的蛋白质毒性。这两种机制在TCF 4突变中仍然没有特征，本项目的成功完成将大大扩展我们对TNR相关FECD发病机制的理解。 规则间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）9核酸酶是在许多实验系统中切割基因组DNA的极其通用和准确的方法。CRISPR/Cas9已被用于使用非同源末端连接（NHEJ）或同源定向修复（HDR）来缺失和替换基因组序列。使用该系统校正FECD中的TCF 4 TNR重复序列尚未进行研究，可能会导致新的治疗方法。 该项目的总体目标是评估1）反义RNA和RAN翻译作为致病机制的作用，以及2）CRISPR/Cas9纠正TCF 4 TNR扩增的潜力。 目的验证TCF 4 TNR扩增后产生的两种效应：1）产生与RNA加工蛋白MBNL 1结合的反义RNA; 2）产生由重复序列相关的非ATG（RAN）翻译产生的异常蛋白。通过使用标记的寡核苷酸和使用荧光原位杂交的MBNL 1评估RNA病灶的共定位来鉴定毒性反义RNA和MBNL 1结合。将通过用编码预测RAN蛋白的质粒转染的转化人角膜内皮细胞的Western印迹和使用针对预测RAN蛋白产生的多克隆抗体的FECD患者内皮组织的免疫组织化学来评估RAN翻译产物。 目的II将检验CRISPR/Cas9可用于纠正TCF 4 TNR扩增的假设。向导RNA（gRNA）将被设计在TCF 4 TNR的任一侧上，并且将被单独评估在293细胞中的切割活性。最好的gRNA将用侧翼寡核苷酸单转染或双转染以评估HDR，并且在没有寡核苷酸的情况下双转染以评估293细胞中的NHEJ，并且随后在具有TCF 4 TNR扩增的转化的FECD角膜内皮细胞中。