Laying a Foundation for Precision Medicine for Fuchs' Dystrophy

项目摘要 Fuchs角膜内皮营养不良（FECD）是一种与年龄相关的退行性疾病，导致角膜水肿 和视力丧失FECD发生在40岁以上的白人中的4%，并且是角膜炎的主要适应症。 超过70%的病例是由TCF 4基因中的CTG三联体重复扩增引起的。 从该基因座表达的扩增的CUG重复RNA（CUGexp）转录物在细胞中积累为核灶。 患者的角膜内皮。CUGexp焦点结合并功能性隔离剪接因子MBNL1 和MBNL2以触发FECD内皮组织中的错误剪接。检测内皮细胞类型特异性 对于FECD，我们将确定FECD患者有丝分裂后角膜内皮细胞的体细胞突变是否会导致 在一个更大的三联体重复扩增比在他们的血液。我们将检查其他眼前节细胞类型 包括角膜上皮细胞、基质角膜细胞和小梁网细胞， CUGexp病灶和相关分子缺陷。为了检验MBNL封存假说，我们将检查，如果 健康供体内皮组织中MBNL的敲低足以重现错误剪接， 在病程早期发现的细胞外基质（ECM）基因的上调。在早期和晚期FECD中，我们 观察到cochlin基因的显著过表达，该基因产生分泌的ECM蛋白， 募集免疫细胞先前在患有青光眼的患者的小梁网中检测到cochlin蛋白， 原发性开角型青光眼（POAG），我们的初步数据表明，该蛋白存在于 FECD受试者的房水和小梁网。我们将检测在小鼠中Cochlin蛋白的水平， 角膜组织，房水样本，和小梁网组织的FECD患者的可能 角膜疾病的结果和增加青光眼的风险在这些患者中的贡献。另外我们 将在我们的UTSW FECD大型队列中检查POAG的患病率，并确定是否存在相关性 三重重复序列长度。我们将使用单细胞RNA测序结合IHC来鉴定免疫 细胞及其对晚期FECD的贡献。我们已经表征了45种反义核酸的功效， 在一些实施方案中，靶向寡核苷酸（ASO）和双链体RNA阻断致病性CUGexp病灶。使用患者来源的细胞 和组织，我们将根据它们阻断病灶形成的能力和它们的特异性来检查它们的效力。 对靶事件和脱靶事件的全转录组评估。我们将检查交付，行动的长度， 使用野生型小鼠的安全性。为了能够进行疾病生物学研究和体内药物测试，我们已经生成了 我们认为这是第一个敲入了扩展CTG重复序列的小鼠模型。在初步研究中，我们 表明我们敲入策略成功地重现了角膜内皮中CUGexp病灶形成。我们将 通过老化这些小鼠以检查转录和组织学特征，进一步表征该鼠模型。 改变FECD疾病，然后使用这些小鼠测试我们的前导CUG重复靶向寡核苷酸， 治疗发展

PROJECT SUMMARY Fuchs’ endothelial corneal dystrophy (FECD) is an age-related degenerative disorder resulting in corneal edema and loss of vision. FECD occurs in 4% of whites over the age of 40 years and is the leading indication for corneal transplantation in the U.S. Over 70% of cases are caused by a CTG triplet repeat expansion in the TCF4 gene. Expanded CUG repeat RNA (CUGexp) transcripts expressed from this gene locus accumulate as nuclear foci in the corneal endothelium of patients. The CUGexp foci bind and functionally sequester the splicing factors MBNL1 and MBNL2 to trigger mis-splicing in FECD endothelial tissue. To examine the endothelial cell-type specificity for FECD, we will determine if somatic mutations in the post-mitotic corneal endothelium of FECD patients results in a larger triplet repeat expansion than in their blood. We will examine if other anterior segment cell-types including corneal epithelium, stromal keratocytes, and trabecular meshwork cells are prone to accumulation of CUGexp foci and associated molecular defects. To test the MBNL sequestration hypothesis, we will examine if the knockdown of MBNLs in healthy donor endothelial tissue is sufficient to recapitulate the mis-splicing and upregulation of extracellular matrix (ECM) genes found early in the disease course. In early and late FECD, we observed a marked overexpression of the cochlin gene that produces a secreted ECM protein also capable of recruiting immune cells. The cochlin protein was previously detected in the trabecular meshwork of patients with primary open angle glaucoma (POAG), and our preliminary data indicate that the protein is present in the aqueous humor and trabecular meshwork of FECD subjects. We will examine levels of the cochlin protein in corneal tissue, aqueous humor samples, and trabecular meshwork tissue of FECD patients for its possible contribution to corneal disease findings and the increased risk for glaucoma in these patients. Additionally, we will examine the prevalence of POAG in our large UTSW FECD cohort and determine if there is a correlation with the triplet repeat length. We will use single-cell RNA sequencing combined with IHC to identify the immune cells and their contribution to late-stage FECD. We have characterized the efficacy of 45 antisense oligonucleotides (ASOs) and duplex RNAs that block disease-causing CUGexp foci. Using patient-derived cells and tissue, we will examine their potency based on their ability to block foci formation and their specificity based on transcriptome-wide assessment of on- and off-target events. We will examine delivery, length of action, and safety using wild-type mice. To enable studies of disease biology and in vivo drug testing, we have generated what we believe is the first mouse model with a knock-in of expanded CTG repeats. In preliminary studies, we show that our knock-in strategy successfully recapitulates CUGexp foci formation in corneal endothelium. We will further characterize this murine model by aging these mice to examine for the transcriptional and histologic alterations of FECD disease, and then use these mice to test our lead CUG-repeat targeting oligonucleotides for therapeutic development.