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

 描述(申请人提供)：Fuchs内皮性角膜营养不良(FECD)以内皮细胞死亡、角膜水肿和视力丧失为特征。FECD影响了大约4%的美国人口。唯一确定的治疗方法是内皮角膜移植，FECD是2013年美国角膜移植的主要适应症。FECD是一种在美国对公共健康有重大影响的角膜疾病，因此与美国国立卫生研究院的使命有关。这种重要的角膜疾病的主要未得到满足的需求是更好的 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TCF4基因的三核苷酸重复(TNR)扩增引起的。TNR扩增的主要致病机制包括1)反义转录的RNA毒性和2)重复相关的非ATG(RAN)翻译的蛋白质毒性。TCF4基因突变的这两种机制尚不明确，该项目的成功完成将极大地扩展我们对TNR相关FECD发病机制的理解。在许多实验系统中，簇状规则间隔短回文重复序列(CRISPR)/CRISPR相关(Cas)9核酸酶是一种非常通用和准确的基因组DNA切割方法。CRISPR/Cas9已被用于使用非同源末端连接(NHEJ)或同源定向修复(HDR)来删除和替换基因组序列。使用该系统纠正FECD中的TCF4 TNR重复还没有被研究过，并可能导致新的治疗方法。本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TCF4 TNR扩张的可能性。目的验证TCF4 TNR扩增产生与RNA加工蛋白结合的反义RNA，肌样蛋白1(MBNL1)和重复序列相关的非ATG(RAN)翻译产生异常蛋白的假设。毒性反义RNA和MBNL1结合将通过荧光原位杂交评估标记的寡核苷酸和MBNL1的RNA灶的共存来鉴定。RAN翻译产物将通过对转化的人角膜内皮细胞进行Western blotting和FECD患者内皮组织的免疫组织化学(使用针对预测的RAN蛋白的多克隆抗体)进行评估。目的II将检验CRISPR/Cas9可用于纠正TCF4 TNR扩张的假设。引导RNA(GRNAs)将设计在TCF4 TNR的两侧，并将单独评估293细胞的切割活性。最好的gRNA将被单或双转染以评估HDR，并在没有寡核苷酸的情况下双转染293细胞以评估NHEJ，随后在TCF4 TNR扩增的转化的FECD角膜内皮细胞中评估NHEJ。

 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.