Gene therapy in the cornea

描述：角膜雾霾和新生血管每年影响超过150万美国人，是全球失明的主要原因之一。基因治疗是预防/治疗这些角膜疾病的一种有吸引力的新方法。然而，由于缺乏组织靶向基因转移方法，基因治疗的临床应用受到严重限制。为了开发针对角膜的组织靶向选择性基因治疗方法，我们假设外源基因可以使用适当的载体和载体传递技术在角膜中选择性地在所需的部位表达，并持续一段时间。用AAV血清型2或5、慢病毒或含有CMV或混合CMV+鸡-肌动蛋白启动子的质粒载体以及定义的载体传递技术进行的初步体内实验表明，转基因可以在体内正常/受损角膜的角质细胞中精确表达所需的时间。Decorin（一种小的富含亮氨酸的蛋白多糖）基因疗法在各种疾病动物模型中已被证明可以预防纤维化和血管生成。这些报道使我们假设，在角质细胞中选择性表达decorin可以抑制/防止角膜模糊和新生血管形成，且副作用最小。为验证这一假设而进行的体外研究表明，decorin能够抑制角化细胞向肌成纤维细胞的转化。已知这种转化会在体内引起角膜雾霭。我们进一步假设，在不改变TGF¿和BIGH3等致病基因在重要器官中的表达的情况下，可以利用选择性组织靶向基因转移方法建立动物模型，在体内研究TGF¿和BIGH3等致病基因在成人角膜中的特定功能。这些基因的功能无法用传统的转基因方法来研究，因为TGF¿缺陷转基因动物会出现致命缺陷，并在4周龄时死亡。本研究的具体目的是验证以下假设：1)载体和载体传递技术调节体内角质细胞中转基因表达的水平、持续时间和位置；2)decorin基因治疗可控制角膜雾霾；3)decorin基因治疗可抑制角膜新生血管形成。使用小鼠模型、测试载体和优化的载体递送技术，我们将测试特异性靶1，并将定义短期和长期的角膜选择性基因转移方法。兔模型将被用来测试特异性目标2和3，通过最佳的选择性基因转移方法将decorin传递到角化细胞。

DESCRIPTION: 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-¿-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 TGF¿ 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 TGF¿-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 approaches for 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.