Mechanisms of Epithelial Alterations in Diabetic Cornea

描述（由申请人提供）：糖尿病眼病是全球失明的主要原因，影响包括角膜在内的所有眼部组织。超过70%的糖尿病患者患有视力下降和疼痛的角膜问题，如神经病变和各种上皮改变，即糖尿病性角膜病变。角膜病变在严重糖尿病中更为明显，但诊断不足；它的治疗仍然是对症的。我们使用足够安全的复制缺陷腺病毒（AV）对器官培养的人角膜进行基因治疗，结果表明：(1)西地那非增强了AV上皮的传递；(2)在糖尿病角膜中，c-met原癌基因上调通过p38激活使特异性蛋白的表达和上皮创面愈合正常化；(3)在正常角膜中，糖尿病角膜组织蛋白酶F和基质金属蛋白酶(MMP)-10升高，可延缓创面愈合，改变标志物对糖尿病的表达；这是通过抑制Akt活性实现的；(4)用AV-shRNA沉默蛋白酶基因后，糖尿病角膜愈合更快。AV基因治疗能够纠正与糖尿病性角膜病变相关的关键上皮异常。我们还表明，几种假定的干细胞标记物在糖尿病角膜缘上皮中的表达降低，使其成为基因治疗的重要靶点。我们的主要假设是糖尿病患者角膜缘上皮的缺陷最终导致角膜上皮异常。这些缺陷可以通过靶向角膜缘上皮细胞的基因治疗来纠正，这是治疗糖尿病性角膜病变的一种很有前途的方法。基于这一假设，我们提出以下具体目标：研究av驱动的角膜缘上皮细胞基因治疗是否逆转糖尿病标志物蛋白表达模式，促进器官培养的糖尿病角膜上皮创面愈合。我们预测，我们的基因治疗将恢复假定的干细胞标志物表达和上皮伤口愈合在糖尿病角膜恢复正常。具体目标2。探讨c-met的过表达和MMP-10和组织蛋白酶F的下调是否能使羊膜上培养的糖尿病角膜缘上皮细胞的标志物表达模式和伤口愈合正常化。我们预测基因治疗将使培养的糖尿病角膜缘细胞正常化，以替代病理改变的糖尿病角膜缘上皮。具体目标3。目的：实现转基因培养角膜缘细胞移植到糖尿病角膜缘区，恢复正常干细胞标志物表达和创面愈合。我们预测基因治疗将使糖尿病角膜缘上皮细胞正常化，因此将它们移植到糖尿病角膜缘区将恢复其正常的伤口愈合和标记蛋白表达。健康相关性：我们通过基因治疗靶向角膜上皮祖细胞生态位的新策略可能为糖尿病性角膜病变提供新的治疗方法，糖尿病性角膜病变是影响大多数糖尿病患者的严重临床问题。

DESCRIPTION (provided by applicant): Diabetic eye disease is the leading cause of blindness worldwide, affecting all eye tissues including the cornea. Over 70% of diabetics suffer from corneal problems that can reduce vision and cause pain, such as neuropathy and various epithelial alterations referred to as diabetic keratopathy. Keratopathy is more pronounced in severe diabetes but is under-diagnosed; its therapy remains symptomatic. Our gene therapy in organ-cultured human corneas using sufficiently safe replication-deficient adenovirus (AV) showed that: (1) Sildenafil enhanced AV epithelial delivery; (2) In diabetic corneas, c-met proto-oncogene upregulation normalized expression of specific proteins and epithelial wound healing via p38 activation; (3) In normal corneas, upregulation of cathepsin F and matrix metalloproteinase (MMP)-10 that are increased in diabetic corneas delayed wound healing and changed marker expression towards diabetic; this occurred via inhibiting Akt activity; (4) When proteinase genes were silenced using AV-shRNA, diabetic corneas healed faster. AV gene therapy was able to correct key epithelial abnormalities associated with diabetic keratopathy. We also show that several presumed stem cell markers had a decreased expression in the diabetic limbal epithelium making it an important target of gene therapy. Our main hypothesis is that deficiencies in the limbal epithelium of diabetic patients ultimately lead to corneal epithelial abnormalities. These defects may be corrected by targeting limbal epithelial cells with gene therapy as a promising approach to the treatment of diabetic keratopathy. Based on this hypothesis, we propose the following Specific Aims: Specific Aim 1. To examine whether AV-driven gene therapy on limbal epithelial cells reverses the diabetic marker protein expression pattern and facilitates epithelial wound healing of organ-cultured diabetic corneas. We predict that our gene therapy will restore presumed stem cell marker expression and epithelial wound healing in diabetic corneas towards normal. Specific Aim 2. To determine if overexpression of c-met and downregulation of MMP-10 and cathepsin F normalizes marker expression pattern and wound healing of diabetic limbal epithelial cells cultured on amniotic membrane. We predict that gene therapy will normalize cultured diabetic limbal cells for their use as substitutes for pathologically altered diabetic limbal epithelium in Aim 3. Specific Aim 3. To achieve transplantation of genetically modified cultured limbal cells to the limbal zone of diabetic corneas in order to restore normal stem cell marker expression and wound healing. We predict that gene therapy will normalize diabetic limbal epithelial cells, so that transplanting them into the limbal region of a diabetic cornea will restore its normal wound healing and marker protein expression. Health relevance: our novel strategy of targeting corneal epithelial progenitor cell niche by gene therapy may lead to a new treatment for diabetic keratopathy, a serious clinical problem affecting the majority of diabetics.