Mechanisms of Epithelial Alterations in Diabetic Cornea

糖尿病性眼病是导致工作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角膜病（屏障功能受损， 伤口愈合、复发性糜烂、上皮性角膜炎等），这会降低视力，引起不适， 痛苦糖尿病性角膜病变诊断不足，治疗仅限于症状控制。我们已经发现 糖尿病角膜和培养的糖尿病角膜上皮干细胞（LESC）标记物的下调 LESC;正常化标志物表达和上皮伤口愈合的器官培养的人糖尿病角膜， 腺病毒介导的携带利姆布斯的LESC基因治疗;设计了一种新的快速剥离人羊膜的方法 HAM上的糖尿病细胞可以移植到清创的 角膜总的来说，我们发现了糖尿病LESC的改变，标准化了它们的标志物表达，校正了它们的表达。 通过基因治疗损害伤口愈合，并能够将它们移植到HAM上裸露的角膜。 我们设计了糖尿病角膜定制的无毒基因治疗载体， 纳米生物缀合物（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 反义寡核苷酸（AON）。我们建议使用这些新的NBCs来安全有效地正常化 糖尿病LESC。糖尿病与稳定的表观遗传变化有关，例如，改变DNA甲基化一种新型 使糖尿病LESC正常化的方法是去除甲基化变化。我们建议采取以下措施， 从糖尿病LESC产生诱导多能干细胞（iPSC）并使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纳米生物缀合物基因疗法和/或通过可移植的 从糖尿病LESC衍生的iPSC获得的功能正常的角膜缘上皮细胞。 具体目标1。利用新型纳米生物复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将被设计成下调糖尿病- 增加组织蛋白酶F和MMP-10，上调糖尿病降低的c-met。 具体目标2。具体目标2。建立一种新的正常化培养糖尿病LESC的方法， 从LESC产生iPSC并将iPSC再分化回角膜缘上皮细胞。我们预测 iPSC会脱落一些糖尿病表观遗传甲基化，使它们转化为正常的角膜缘细胞。 具体目标3。具体目标3。为了实现纳米药物治疗和糖尿病iPSC的移植， HAM上的衍生角膜缘细胞对糖尿病角膜和正常干细胞的恢复比较 标志物表达和伤口愈合。将比较iPSC和纳米基因疗法的功效。 我们的目标符合NEI Vision Research的优先事项：改进培养的角膜上皮移植 细胞，了解伤口愈合的表观遗传学，并开发方法，以提高伤口愈合。

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.