Gene therapy in the cornea

角膜雾霾和新生血管每年影响超过150万美国人，是 世界范围内导致失明的主要原因。基因治疗是一种诱人的新的预防/治疗方法。 这些角膜疾病。然而，由于基因治疗的不可获得性，其临床应用受到严重限制。 组织靶向基因转移方法。开发组织靶向的选择性基因治疗方法 对于角膜，我们假设外源基因可以在所需的时间选择性地在角膜中表达 在选定的持续时间内，使用适当的载体和载体传递技术。体内初步研究 用AAV血清2或5型、慢病毒或含有CMV或杂交病毒的质粒载体进行的实验 CMV+鸡-3-肌动蛋白启动子，以及明确的载体传递技术证明，转基因可以 在体内正常/受损角膜的角质形成细胞中精确表达所需时间。装饰 (一种富含亮氨酸的小蛋白多糖)基因治疗已被证明可以预防糖尿病患者的纤维化和血管生成 各种疾病动物模型。这些报告使我们推测，核心蛋白的选择性表达 角膜基质细胞可以抑制/防止角膜混浊和新生血管，副作用最小。试管苗 用来检验这一假说的研究证实了核心蛋白聚糖抑制角质形成细胞的能力。 转化为肌成纤维细胞。这种转变在活体内会导致角膜混浊。我们进一步 假设选择性组织靶向基因转移方法可用于建立动物模型 为了研究TGFp和BIGH3等致病基因在成人角膜中的特定功能 在不改变它们在重要器官中的表达的情况下，在体内表达。这类基因的功能不能用 传统的转基因方法，因为缺乏TGFp的转基因动物存在致命的缺陷和 在4周大的时候死亡。检验假设的具体目的是1)向量和向量传递技术 在活体角质形成细胞中调节转基因表达的水平、持续时间和位置2)核心蛋白聚糖基因治疗 3)核心蛋白聚糖基因治疗可抑制角膜新生血管。使用鼠标 模型、测试的矢量和优化的矢量传递技术，我们将测试特定的目标1并将定义 针对角膜的短期和长期选择性基因转移方法。将使用兔子模型来测试 通过最佳的选择性基因转移方法将核心蛋白聚糖导入角质形成细胞，从而获得特异性靶点2和3。

Corneal haze and neovascularization affect over 1.5 million Americans every year and are among the leading causes of blindness worldwide. Gene therapy is an attractive and novel approach to prevent/treat these corneal disorders. However, clinical utility of gene therapy is severely limited due to unavailability of tissue-targeted gene transfer methods. To develop tissue-targeted selective gene therapy approaches for the cornea, we hypothesized that foreign genes can be selectively expressed in the cornea at the desired site for selected duration using appropriate vectors and vector-delivery techniques. Preliminary in vivo experiments performed with AAV serotype 2 or 5, lentivirus or plasmid vectors containing CMV or hybrid CMV+chicken-3-actin promoter, and defined vector-delivery techniques demonstrated that transgene can be precisely expressed in keratocytes of normal/damaged corneas in vivo for the desired time period. Decorin (a small leucine-rich proteoglycan) gene therapy has been shown to prevent fibrosis and angiogenesis in various disease animal models. These reports led us to hypothesize that selective expression of decorin in keratocytes can inhibit/prevent corneal haze and neovascularization with minimal side effects. The in vitro studies performed to test this hypothesis demonstrated competence of decorin to inhibit keratocyte transformation to myofibroblasts. This transformation is known to cause corneal haze in vivo. We further hypothesize that selective tissue-targeted gene transfer approaches can be used to develop animal models for studying the specific function of disease-causing genes such as TGFp and BIGH3 in the adult cornea in vivo without altering their expression in vital organs. The function of such genes cannot be studied using conventional transgenic approaches because TGFp-deficient transgenic animals suffer lethal defects and die by 4 weeks of age. The specific aims to test the hypotheses are 1) vector and vector-delivery techniques regulate level, duration, and location of transgene expression in keratocytes in vivo 2) decorin gene therapy can control corneal haze and 3) decorin gene therapy can inhibit corneal neovascularization. Using a mouse model, tested vectors, and optimized vector delivery-techniques, we will test Specific Aim 1 and will define short- and long-term selective gene transfer approachesfor the cornea. Rabbit models will be used to test Specific Aims 2 and 3 by delivering decorin into keratocytes with optimal selective gene transfer methods.