Mechanisms of epithelial alterations in diabetic cornea

糖尿病是工作年龄成年人中最普遍的致盲疾病。高达70%的糖尿病患者 角膜问题，包括神经病变和上皮角膜病变（伤口愈合延迟，复发性 糜烂、溃疡），损害视力并引起疼痛和不适。糖尿病性角膜病变诊断不足， 治疗仍然是对症治疗我们确定了人类糖尿病角膜中改变的标记物，并恢复了它们的 通过基因和纳米治疗，角膜器官培养物中的正常水平，这也恢复了正常的干细胞 表型和角膜伤口愈合。表观遗传变化似乎也有助于糖尿病并发症。 在上一个资助期，我们设计了新的方法来使糖尿病角膜正常化（1），以避免毒性 和（2）消除糖尿病细胞中稳定的表观遗传变化。我们开发了无毒的纳米生物复合物 (NBC)在培养的LEC和器官中有效地使干细胞标志物表达和伤口愈合正常化， 培养的角膜，基于c-Met、MMP-10和组织蛋白酶F表达的反义调节。我们也 发现了一种新的标记，Wnt 5a，通过双重DNA甲基化和microRNA-203减少糖尿病角膜 表观遗传抑制向糖尿病细胞或角膜添加Wnt 5a或去甲基化剂地西他滨 恢复伤口愈合动力学和干细胞标志物表达。在另一种方法中，我们产生了诱导 来自糖尿病LEC的多能干细胞（iPSC）以去除糖尿病表观遗传特征并分化 使它们恢复到可能正常化的角膜缘样上皮。这些以前的糖尿病细胞表达几个干细胞。 细胞标志物和我们预期的一样在这个建议中，我们将优化人类的正常化， 糖尿病角膜在结构上（通过干细胞标志物表达）和功能上（通过伤口愈合动力学）。 我们假设（1）靶向几种糖尿病标志物的NBC与DNA去甲基化的组合， 药物将提供比单一疗法更有效的糖尿病角膜正常化，和（2）iPSC衍生的 采用气提法和自然培养法可将LEC样细胞定向分化为可移植的角膜上皮细胞 角膜表面支持。我们的目标是从机制上了解正常化治疗如何改变基因， 和蛋白质表达水平，并影响角膜细胞群，使用经验证的单细胞RNA-seq方法。 目标1。开发纳米和表观遗传学联合治疗糖尿病角膜伤口愈合和祖细胞 细胞使用新的NBCs和DNA去甲基化剂。目标2.为了解开基因表达的变化， 在用组合的抗糖尿病药物治疗后通过scRNA-seq测定的糖尿病角膜上皮中的细胞群体 药理学试剂。目标3。为了产生和表征具有非- 在使用糖尿病iPSC衍生的角膜缘样上皮细胞的气升培养物中的糖尿病性质。 我们的目标非常符合NEI战略计划2021的主要举措：（1）改变游戏规则的进步-获得 转录组学分析中的单细胞分辨率，（2）探索不同的细胞疗法和组织模型 （3）优化用于眼睛的基因递送，包括非病毒系统，如纳米颗粒。

Diabetes is the most widespread blinding disease in working-age adults. Up to 70% of diabetic patients suffer from corneal problems including neuropathy and epithelial keratopathy (delayed wound healing, recurrent erosions, ulcers) that impair vision and cause pain and discomfort. Diabetic keratopathy is underdiagnosed, and therapy remains symptomatic. We identified markers altered in human diabetic corneas and restored their normal-like levels in corneal organ cultures by gene and nano therapy, which also restored normal stem cell phenotype and corneal wound healing. Epigenetic changes also appear to contribute to diabetic complications. In the previous funding period, we designed new approaches to normalize diabetic corneas (1) to avoid toxicity and (2) to remove stable epigenetic changes in diabetic cells. We developed nontoxic nanobioconjugates (NBC) that efficiently normalized stem cell marker expression and wound healing in cultured LEC and organ- cultured corneas, based on antisense modulation of c-Met, MMP-10, and cathepsin F expression. We also discovered a new marker, Wnt5a, reduced in diabetic corneas by dual DNA methylation and microRNA-203 epigenetic suppression. Addition of Wnt5a or demethylating agent Decitabine to diabetic cells or corneas restored wound healing kinetics and stem cell marker expression. In another approach, we produced induced pluripotent stem cells (iPSC) from diabetic LEC to remove diabetic epigenetic signatures and differentiated them back to potentially normalized limbal-like epithelium. Such formerly diabetic cells expressed several stem cell markers similar to normal, as we expected. In this proposal, we will optimize normalization of human diabetic corneas structurally (by stem cell marker expression) and functionally (by wound healing kinetics). We hypothesize that (1) a combination of NBC targeting several diabetic markers with DNA demethylating agents would provide more efficient diabetic corneal normalization than single therapies, and (2) iPSC-derived LEC-like cells could be differentiated into transplantable corneal epithelial cells using air-lifting and natural corneal surface support. We aim to understand mechanistically how the normalizing treatments change gene and protein expression levels and affect corneal cell populations using validated single-cell RNA-seq approach. Aim 1. To develop combined nano and epigenetic therapy for diabetic corneal wound healing and progenitor cells using novel NBCs and DNA demethylating agents. Aim 2. To unravel changes in gene expression and cell populations in diabetic corneal epithelium by scRNA-seq following treatment with combined pharmacological agents. Aim 3. To produce and characterize differentiated corneal epithelium with non- diabetic properties in air-lifted cultures using diabetic iPSC-derived limbal-like epithelial cells. Our aims fit well major initiatives of the NEI Strategic Plan 2021: (1) A game-changing advance – gaining single-cell resolution in transcriptomic analyses, (2) Explore different cell therapies and tissue models derived from iPSCs, and (3) Optimize gene delivery for the eye including non-viral systems, such as nanoparticles.