Targeted Gene Therapy and Nanomedicine Approaches to Treat Corneal Diseases

描述（由申请人提供）： 摘要眼睛的损伤和感染引起角膜瘢痕，每年影响超过150万美国人。尽管使用了战斗眼睛保护，但我们在阿富汗和伊拉克的受伤士兵中有16%发生了眼睛受伤。目前用于角膜瘢痕形成或血管生成的疗法仅提供短期缓解，引起严重的副作用并且通常无效。美国每年有超过5万例角膜移植手术，在最近的战争中受伤的美国军队中有20%的眼睛被摘除，这突出了目前治疗的不足。角膜是基因治疗的理想器官，因为它的可及性和免疫豁免状态。最近，我们定义了用于正常和患病兔眼的组织靶向GNP或AAV 5载体基因治疗方法，并表明治疗基因可以有效地递送到靶角膜细胞中以治疗角膜瘢痕形成和血管生成。我们最近的研究表明，金纳米颗粒（GNP）对角膜是安全的，因此可以用于在体内角膜中传递基因。我们的中心假设是，角膜中的组织选择性治疗性基因递送可以治愈角膜疾病而不会引起显著的副作用。该研究项目将测试以下假设：稳定在阿拉伯树胶或聚乙烯亚胺中的GNP是无毒、无免疫原性和有效的载体，用于将治疗基因转运到兔角膜的角膜细胞中，可以提供安全和新颖的基于纳米技术的非病毒基因治疗模式来治疗角膜疾病，并且靶向递送基因使转化生长因子-2（TGF-2）功能失效将阻止体内角膜瘢痕形成。TGF 2基因已显示通过促进角膜细胞转化为肌成纤维细胞而在体内引起角膜瘢痕形成。我们将定义使用稳定在阿拉伯树胶或聚乙烯亚胺中的GNP（Aim 1）将治疗性基因递送到兔角膜细胞中的条件，通过使用GNP或AAV 5载体将选定的基因递送到兔角膜细胞中来测试可溶性II型TGF 2受体（Aim 2）和骨形态发生蛋白7（Aim 3）基因控制角膜瘢痕形成的功效。我们的初步研究表明，测试的GNP和AAV 5载体在体内将基因选择性导入兔角膜细胞方面非常有效。利用兔角膜瘢痕模型，组织靶向的基因治疗方法（最近在我们的实验室优化），生物显微镜，免疫细胞化学，细胞和分子检测测试这些目标将完成。成功完成拟议的研究有可能扩展一种有效的新方法来治疗角膜瘢痕以及其他流行的角膜疾病。我们对GNP的研究将极大地推动眼纳米医学领域的研究工作。 公共卫生相关性： 项目叙述：与退伍军人健康的相关性临床导向的基础科学研究是重要的，并实现了退伍军人健康管理局的目标，为美国退伍军人实现卓越的医疗保健。这一建议提供了一个推动力的发展，组织选择性基因治疗方法，使用金纳米粒子治疗角膜瘢痕，提供长期的好处。现有的治疗方法只能提供短期的益处，而且往往无效。拟议的临床前研究为急需的靶向，有效和长期治疗角膜瘢痕的发展奠定了基础。已知眼睛的损伤和感染会导致角膜瘢痕形成。在阿富汗和伊拉克战争中，我们大约有16%的受伤士兵尽管戴着战斗眼睛保护装置，但眼睛仍然受伤。眼外伤导致的视力丧失是我们军队中的一个主要问题，目前没有药物可以治疗角膜瘢痕而不引起严重的副作用。

DESCRIPTION (provided by applicant): Abstract Injury and infection to the eye cause corneal scarring and affect over 1.5 million Americans each year. Despite the use of combat eye protection, eye injuries occurred in 16% of our wounded troops in Afghanistan and Iraq. Current therapies for corneal scarring or angiogenesis provide only short-term relief, cause serious side effects and are often ineffective. Over fifty thousand corneal transplantations in Americans each year and removal of eyes in 20% of the U.S. troops injured in recent wars highlight the incompetence of current therapy. The cornea is an ideal organ for gene therapy because of its accessibility and immune-privilege status. Recently we defined tissue-targeted GNPs or AAV5 vector gene therapy approaches for the normal and diseased rabbit eye, and showed that therapeutic genes can be delivered into target corneal cells efficiently to treat corneal scarring and angiogenesis. Our recent studies suggest that gold nanoparticles (GNPs) are safe for the cornea and thus can be used to deliver genes in the cornea in vivo. Our central hypothesis is that tissue-selective therapeutic gene delivery in the cornea can cure corneal disorders without causing significant side effects. This research project will test the hypotheses that GNPs stabilized in gum arabic or polyethyleneimine are nontoxic, non-immunogenic and efficient vehicles for transporting therapeutic genes into keratocytes of the rabbit cornea in vivo, can provide safe and novel nanotechnology-based nonviral gene therapy modalities to treat corneal diseases, and targeted delivery genes disabling transforming growth factor-2 (TGF2) function will impede corneal scarring in vivo. TGF2 gene has been shown to cause scarring in the cornea in vivo by promoting keratocyte transformation to myofibroblasts. We will define conditions for delivering therapeutic genes into rabbit keratocytes in vivo using GNPs stabilized in gum arabic or polyethyleneimine (Aim 1), test the efficacy of soluble type II TGF2 receptor (Aim 2) and bone morphogenic protein 7 (Aim 3) genes to control corneal scarring by delivering selected genes into rabbit keratocytes in vivo with GNPs or AAV5 vector. Our preliminary studies show that tested GNPs and AAV5 vectors are highly efficient in introducing genes selectively into rabbit keratocyte in vivo. Using rabbit corneal scarring model, tissue-targeted gene therapy approaches (recently optimized in our laboratory), bio-microscopy, immunocytochemistry, cellular and molecular assays testing of these aims will be accomplished. Successful completion of proposed research has the potential to extend an efficient new method to treat corneal scarring as well as other prevalent corneal diseases. Our studies on GNPs will substantially advance the research efforts in the field of ocular nanomedicine. PUBLIC HEALTH RELEVANCE: Project Narrative: Relevance to Veterans Health Clinically-oriented basic science research is important and fulfills the objective of the Veterans Health Administration objective to achieve excellence in health care for American veterans. This proposal provides an impetus toward the development of tissue-selective gene therapy methods using gold nanoparticles to treat corneal scarring which provide long-term benefits. Existing therapies provide only short-term benefits and are often ineffective. The proposed preclinical research lays the groundwork for the much-needed development of targeted, effective and long-term therapies to cure corneal scarring. Injury and infections to eye is known to cause corneal scarring. About 16% of our wounded troops in Afghanistan and Iraq wars sustained eye injuries despite wearing combat eye protection. Vision loss from eye trauma is a major problem among our military and no drugs are currently available to cure corneal scarring without causing serious side effects.