Laying a Foundation for Precision Medicine for Fuchs' Dystrophy

项目总结 Fuchs内皮性角膜营养不良(FECD)是一种与年龄相关的退行性疾病，导致角膜水肿 和失明。FECD发生在4%的40岁以上的白人身上，是角膜的主要适应症 在美国，超过70%的移植病例是由TCF4基因的CTG三联体重复扩增引起的。 从该基因位点表达的扩展CUG重复RNA(CUGexp)转录本在 患者的角膜内皮。CUGexp焦点结合和功能隔离剪接因子MBNL1 和MBNL2在FECD内皮组织中触发错误剪接。检测内皮细胞类型的特异性 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 在一个更大的三胞胎中重复扩张比在他们的血液中。我们将检查其他眼前节细胞类型 包括角膜上皮、基质角质形成细胞和小梁细胞，容易积聚 CUGexp焦点及其相关的分子缺陷。为了检验MBNL隔离假说，我们将检验 在健康的供体内皮组织中MBNLS的敲除足以概括错误剪接和 在病程早期发现的细胞外基质(ECM)基因上调。在FECD早期和晚期，我们 观察到显著过度表达的cochlin基因，它产生一种分泌的ECM蛋白，也能够 招募免疫细胞。先前在小梁网中检测到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受试者房水和小梁网。我们将检测柯克林蛋白的水平。 FECD患者角膜组织、房水标本和小梁组织的检测 对这些患者的角膜疾病发现和青光眼风险增加的贡献。此外，我们 将检查我们的大量UTSW FECD队列中POAG的患病率，并确定是否存在相关性 具有三联体重复长度。我们将使用单细胞RNA测序和免疫组化相结合的方法来鉴定免疫 细胞及其在晚期FECD中的作用。我们已经确定了45个反义基因的有效性。 阻断致病CUGexp焦点的寡核苷酸(ASO)和双链RNA。使用患者来源的细胞 和组织，我们将根据它们阻止病灶形成的能力和它们的特异性来检查它们的效力 在转录组范围内评估目标上和目标外的事件。我们将检查交货、行动期限和 使用野生型小鼠安全。为了能够进行疾病生物学和体内药物测试的研究，我们已经生成了 我们认为这是第一个带有扩展的CTG重复序列的小鼠模型。在初步研究中，我们 结果表明，我们的敲入策略成功地概括了角膜内皮中CUGexp焦点的形成。我们会 通过老化这些小鼠以检查转录和组织学来进一步表征这个小鼠模型 FECD疾病的改变，然后使用这些小鼠来测试我们的领先的CUG-Repeat靶向寡核苷酸 治疗方面的发展。

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.