Mechanisms of Epithelial Alterations in Diabetic Cornea

糖尿病眼病是劳动年龄成年人失明的主要原因。超过70%的糖尿病患者 患有角膜问题，主要是神经病和上皮性角膜病变(屏障功能受损和 伤口愈合、反复腐烂、上皮性角膜炎等)，会降低视力，引起不适和 疼痛。糖尿病角膜病变被低估，治疗仅限于症状控制。我们发现 糖尿病角膜和培养糖尿病患者角膜缘上皮干细胞标志物的下调 归一化标记物表达与器官培养的人糖尿病角膜上皮损伤愈合 携带LESC的角膜缘腺病毒基因治疗设计了一种新的快速剥离羊膜的方法 用于细胞生长的膜(HAM)；显示HAM上的糖尿病细胞可以移植到清创者身上 眼角膜。总体而言，我们发现了糖尿病LESCs的变化，正常化了它们的标志物表达，纠正了 通过基因治疗损害伤口愈合，并能够将它们移植到裸露的角膜上。 我们设计了专为糖尿病角膜量身定做的无毒基因治疗载体，以天然衍生的聚马来酸为基础 纳米生物结合物(NBC)。它们通过附着的抗体与靶细胞结合，并调节特定的标志物 用反义寡核苷酸(AON)。我们建议使用这些新的NBC来安全有效地实现正常化 糖尿病LESC。糖尿病与稳定的表观遗传学改变有关，例如DNA甲基化改变。一本小说 使糖尿病LESC正常化的方法是消除甲基化变化。我们建议通过以下方式实现这一目标 从糖尿病LESC生成诱导多能干细胞(IPSC)，并将IPSC分化为角膜缘细胞。 我们的主要假设是糖尿病角膜缘上皮干细胞的正常化和伤口愈合 可以通过使用新的无毒的纳米生物偶联基因疗法和/或通过可移植的方式实现 从糖尿病LESC来源的IPSCs中获得功能正常的角膜缘上皮细胞。 具体目的1.利用新型纳米生物结合物开发一种安全的糖尿病LESC基因治疗方法 通过反义抑制调节糖尿病标志物。NBC将被设计来降低糖尿病的水平- 升高组织蛋白酶F和基质金属蛋白酶-10，上调糖尿病，降低c-Met。 特殊目的2.特殊目的2.发展一种使培养的糖尿病LESC正常化的新方法 从LESC产生IPSC，并将IPSC重新分化为角膜缘上皮细胞。我们预测 这种IPSC可以去除一些糖尿病表观遗传的甲基化，使它们能够转化为正常的角膜缘细胞。 特定目标3.特定目标3.实现纳米治疗和糖尿病IPSC的移植- 角膜缘干细胞移植在糖尿病角膜上的应用及正常干细胞修复的比较 标记物表达与创面愈合IPSC和纳米基因疗法的疗效将进行比较。 我们的目标符合NEI Vision Research的优先事项：改进培养的角膜上皮细胞移植 细胞，了解伤口愈合的表观遗传学，以及开发促进伤口愈合的方法。

Diabetic eye disease is a leading cause of blindness in working-age adults. More than 70% of diabetic patients suffer from corneal problems, mainly from neuropathy and epithelial keratopathy (impaired barrier function and wound healing, recurrent erosions, epithelial keratitis, etc.), which can reduce vision, and cause discomfort and pain. Diabetic keratopathy is underdiagnosed, and therapy is limited to symptom control. We have discovered downregulation of putative limbal epithelial stem cells (LESC) markers in diabetic corneas and cultured diabetic LESC; normalized marker expression and epithelial wound healing in organ-cultured human diabetic corneas by adenoviral gene therapy of LESC-harboring limbus; designed a new fast method of denuding human amniotic membrane (HAM) for cell growth; showed that diabetic cells on HAM could be transplanted to the debrided corneas. Overall, we uncovered alterations in diabetic LESCs, normalized their marker expression, corrected impaired wound healing by gene therapy, and were able to transplant them on HAM to denuded corneas. We designed diabetic cornea-tailored non-toxic gene therapy vehicles, natural-derived polymalic acid-based nanobioconjugates (NBC). They bind to targeted cells by attached antibodies and modulate specific markers with antisense oligonucleotides (AON). We propose to use these new NBCs to safely and efficiently normalize diabetic LESC. Diabetes is associated with stable epigenetic changes, e.g., altered DNA methylation. A novel way of normalizing diabetic LESC would be to remove methylation changes. We propose to achieve this by generating induced pluripotent stem cells (iPSC) from diabetic LESC and differentiating iPSCs to limbal cells. Our main hypothesis is that normalization of diabetic limbal epithelial stem cells and wound healing could be achieved by using new non-toxic nanobioconjugate gene therapy and/or by transplantable functionally normal limbal epithelial cells obtained from diabetic LESC-derived iPSCs. Specific Aim 1. To develop a safe gene therapy for diabetic LESC using novel nanobioconjugates modulating diabetic markers by antisense inhibition. The NBCs will be designed to downregulate diabetes- increased cathepsin F and MMP-10, and upregulate diabetes-decreased c-met. Specific Aim 2. Specific Aim 2. To develop a novel way of normalizing cultured diabetic LESC by generating iPSC from LESC and redifferentiating the iPSCs back to limbal epithelial cells. We predict that iPSC would shed some diabetic epigenetic methylation, allowing to convert them to normal limbal cells. Specific Aim 3. Specific Aim 3. To achieve transplantation of nanodrug-treated and of diabetic iPSC- derived limbal cells on HAM on the diabetic corneas and compare restoration of normal stem cell marker expression and wound healing. iPSC and nano gene therapy will be compared for efficacy. Our aims fit the priorities of NEI Vision Research: improving transplantation of cultured corneal epithelial cells, understanding epigenetics of wound healing, and developing methods to enhance wound healing.